Low Dose Effects of Pesticides in the Aquatic Environment - ACS

Chapter 12

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Low Dose Effects of Pesticides in the Aquatic Environment Nina Cedergreen*,1 and Jes J. Rasmussen2 1Department

of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark 2Department of Bioscience, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark *E-mail: [email protected]. Telephone: +45 35 33 33 97.

In contrast to the terrestrial environment, organisms in the aquatic environment are exposed to more complex mixtures of pesticides with high concentrations occurring in pulses associated with spray and rain events. To quantify the effect of these complex mixtures, mixture toxicity models have to be used. The standard is to use Concentration Addition (also called Dose Addition), where all co-occurring pesticides are transformed into one common unit, which is summed and used as the joint exposure concentration. Using this approach, both herbicides and insecticides can reach joint concentrations during storm flow that can cause measurable effects in the plant, algae and invertebrate communities. Fungicide concentrations rarely reach the same level of predicted environmental effects, but this is likely owed to the fact that the traditional ecotoxicological tests do not reflect aquatic fungi communities, which are likely the most sensitive to these pesticides. Except for extreme incidents, pesticide occurrences in agricultural catchments will rarely lead to acute extermination of the majority of species. There is no doubt, however, that peak pesticide concentrations can affect the species communities on sub-lethal endpoints such as growth, emergence times, feeding and drift behaviour which ultimately may translate into altered community structure and function. In addition to the low dose effects of pesticides, other stressors of both chemical and physical nature are also stressing © 2017 American Chemical Society Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the aquatic communities. Therefore, ensuring a diverse aquatic community, requires a broad focus including both combatting excessive concentrations of pesticides and other chemicals and improving the physical habitat on a local scale as well as on a catchment scale. Only holistic approaches, including all stressors of the aquatic ecosystem, will sufficiently safeguard aquatic ecosystems and potentially recreate lost habitats, as well as increase populations of threatened species.

Introduction Pesticides used for plant protection, vector control and urban use often end up in the aquatic environment, where they can be detected in both water and sediments. The primary transport routes for pesticides to surface waters in agricultural catchments are surface runoff and tile drainage (1–4). The magnitude of pesticide transport is governed by different climatic and geological factors (e.g. amount and intensity of rainfall, hydrology, field slopes and soil types). In vertical crops such as vine and fruit plantations which are intensively sprayed, spray drift can also be an important route of pesticide transport (5). Pesticide occurrences in urban catchments are mainly related to the size of paved areas (6). Often the pesticide concentrations occurring in surface waters are low, being in the ng/L range. Depending on the physico-chemical properties of the pesticides they will sorb to sediments and/or be degraded within days to weeks or months. A wide range of pesticides is used in conventional agriculture and in urban environments (active ingredients primarily used as biocides) facilitating the possible transport to surface waters. Hence, aquatic communities may be exposed to a broad array of different mixtures of varying complexity. In this chapter, we discuss the occurrence and possible environmental effects of these low doses of pesticides on aquatic communities based on the research we have been involved in, within the past ten to fifteen years.

Characterizing Exposure There is a clear link between the applied quantity and application frequency of pesticides in surface water catchments and the magnitude and occurrence frequency of detectable concentrations of the same pesticides in the adjacent surface water bodies (7, 8). Consequently, pesticides are more often detected in surface waters located in agricultural and urban catchments compared to uncultivated catchments, and countries with higher environmental regulatory requirements for pesticide usage are generally characterized by lower pesticide pollution compared to countries with less strict regulatory requirements (9). However, pesticide concentrations in surface waters are in general characterized by substantial variation in time and space governed in part by climatic factors (especially rain events) and differing seasonal usage, generating a discontinuous 168 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


and complex occurrence pattern in receiving surface waters (1, 10) (Figure 1). Herbicides and fungicides have relatively slow modes of action in both target and non-target organisms and are used during most of the growing season in temperate climate agriculture, which will be the focus of this paper. In contrast, insecticides often have rapid modes of action and target pest organisms that occur in more narrow temporal windows (11).

Figure 1. An example of the variability of pesticide concentrations over time in a small Danish stream, sampled by event triggered sampling. Data were collected by the Danish National Environmental Research Institute and details of sampling is published in Kronvang et al. (87). The data presented are the three herbicides occurring at the highest concentrations from the Nedstrøms, Lillebæk measuring station. In addition to these three herbicides, another ten herbicides and fungicides were also detected following a similar pattern of occurrence peaks during the growth season following rain events. Insecticides were not monitored for in this study. Data were kindly provided by Merete Styczen.

Hence, herbicides and fungicides are generally characterized by higher application frequencies (in part prophylactic treatments), whereas insecticides are generally characterized by comparably lower application frequencies specifically timed with the co-occurrence of insect pest outbreaks. These inherent differences in application patterns between pesticide groups lead to some generic differences in occurrence patterns in surface waters with herbicides and fungicides being more or less continuously detectable in agricultural streams during the crop growing seasons and occurrence of insecticides being rather restricted to the temporal window containing pest insect outbreaks (12–14). 169 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Multiple studies have documented that pesticide concentrations increase in water bodies during precipitation events, with storm water concentrations exceeding those of base flow conditions by a factor of 10-100 (4, 5, 12, 13, 15). The magnitude of concentration increase depends on a series of geological (e.g. soil type), topographic (e.g. slope of soil surface adjacent to the water body) and climatic factors (e.g. precipitation depth and frequency of significant rain events) (see review by Schulz, 2004 (5)). Moreover, the physicochemical properties of pesticides are of significance with more water soluble pesticides being more prone to baseflow leaching. Pesticides of low water solubility, on the other hand, are being associated with stronger but shorter increases in concentrations in receiving water bodies, probably due to their association with small washed-out particles (e.g. Kronvang et al., 2004 (16)). Consequently, in agricultural and urban landscapes the vast majority of the seasonal sum of pesticide flux from catchments to streams occurs during rain events (7). As the acute effect of pesticides in the aquatic environment is most often associated with the peak concentrations, it is worth noting that small water bodies are characterized by higher pesticide concentrations compared to larger water bodies, most likely due to higher connectivity between land and water and a lower dilution potential in the smallest water bodies (17). Larger streams are, on the other hand, more often dominated by larger numbers of different pesticides collected from the larger catchment area. Pesticides and residues occurring in surface waters will, strongly depending on the physicochemical properties of the pesticides, partition between water and organic and inorganic surfaces in the aquatic environment (18–20). The vast majority of pesticides with high Kow (e.g. pyrethroid insecticides) will rapidly adsorb to such organic surfaces and dissipate from the water phase (21). The relative adsorption potential is generally higher for the smallest of sediment particles (18), meaning that the majority of pesticides adsorbed to sediment particles reside in the upper few mm of sediments which additionally is the most mobile fraction (22). Pesticide half-life may be significantly prolonged when adsorbed to aquatic sediments and may therefore serve as a fingerprint of both past and present use (4, 23). The concentration ranges detected are usually in the ng/L range, but can get into the low µg/L range during storm flow and during spray seasons in heavily sprayed areas (4, 7, 12, 17, 24). We have given examples of base flow and storm flow concentrations from a Danish and Californian study in Table 1, representing cultivated catchments and have included an example from measurements in German orchard ditches, which we consider as being representative for very intense peak concentrations.

170 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Country Location description (n sampling sites)

Base flow

Storm flow

n

Mean±stdev (µg/L)

Median (µg/L)

Detected pesticides

n

Mean±stdev (µg/L)

Median (µg/L)

Detected pesticides

Agricultural catchment (n = 10)

10

0.192±0.099

0.077

7.10±3.96

37

1.845±0.339

1.001

21.29±8.43

Control catchments (n = 9)

9

0.033±0.014

0.015

5.00±5.83

28

0.277±0.088

0.075

7.74±4.66

12

3.505±5.11

1.375

-

12

13.91±15.15

6.84

-

Pajaro estuary (n = 4)

37

0.037±0.059

0.018

4.16±3.43

20

0.447±2.691

0.054

9.70±2.27

Salinas estuary (n = 4)

32

0.022±0.033

0.013

4.31±1.92

16

0.140±0.476

0.041

6.56±3.32

Santa Maria estuary (n = 4)

27

0.273±1.016

0.058

10.3±2.2

12

0.239±0.330

0.112

12.4±6.1

Denmarka

171


Table 1. Examples of Concentration Ranges of Representative Pesticides Measured in Surface Waters under Base Flow and Storm Flow Events in Three Countries with Different Topography and Agricultural Practices. the Data Are from Rasmussen et al. (4), Lorenz et al. (17) and Smalling and Orlando (12). For All Catchments the Number of Water Samples Measured, the Mean Concentrations of All Measured Pesticides > Limit of Detection (LOD), the Median Concentration and the Number of Pesticides Detected >LOD in Each Sample Are Given ±stdev.

Germanyb Orchard ditches (n = 2) Californiac

The Danish base flow samples were collected in August 2012 in 19 streams of agricultural dominated catchment and control catchments. The storm flow samples were collected by event triggered sampling in the period May-June 2012, and 70 pesticides and pesticide metabolites were analyzed for. b The German study investigated the difference between weekly integrated samples (base flow) and samples taken within 1-2 days after a spray event (“storm flow”) in ditches in an orchard region. Results from six pesticides were reported. c The Californian study investigated three catchments during 4 event triggered storm flow events during 2008 and 2009, including 11 samplings during base flow. They monitored 68 pesticides and pesticide metabolites. The data was kindly provided by Kelly Smalling. a

Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


How Do We Deal with Mixtures? As mentioned above, the exposure scenario in the aquatic environment is characterized by its high complexity where co-exposure to at least 10-20 pesticides is the rule rather than the exception (3, 4, 25). Within the field of aquatic toxicology there has therefore been a large focus on how to predict the joint effect of mixtures. Basically there are two competing concepts for predicting mixture effects: Concentration Addition (also called Dose Addition), assuming that the compounds have a similar mode of action, and Independent Action (also called Response Multiplication, Response Addition and Effect Addition), assuming binary endpoints and dissimilar mode of action (For a review of the concepts and their use, see Cedergreen et al, 2013 (26)). A range of studies performed during the past two decades have shown Concentration Addition (CA) to explain binary mixtures of pesticides within a two-fold error for approximately 90% of the tested mixtures, disregarding the mode of action of the pesticide combination (27, 28). Also, comparing the two concepts showed relatively little difference between model predictions for a range of test systems, with CA most often being the conservative model (29). In addition, several studies have shown that increasing the number of chemicals in a mixture usually decrease the deviation from the reference model (30, 31). Hence, for risk assessment purposes, there is broad agreement concerning the use of CA as a reference model. The basic concept of CA is that all chemicals act in a similar way, and less toxic chemicals simply act as a dilution of the more potent chemicals. If you have a mixture, you can therefore convert all chemical concentrations to the same unit, the toxic unit (TU), by dividing their concentration by an EC-value from a specific test. Often the EC50 of the species believed to be most sensitive is used. For joint risk assessment of pesticides, using CA as a reference model is particularly convenient, as there are available EC-values for at least algae, daphnids and fish for all registered compounds. The toxic units of a specific mixture are then summed up (∑TU), giving a measure of the joint concentration of the mixture. Benchmark concentrations for ∑TU for different groups of organisms have been found (32), and the risk of a particular water sample toward the aquatic community can then be evaluated. Importantly also, using the toxic unit principle opens the possibility of investigating which chemicals in the mixture contributes the most to the joint toxicity. This is done by investigating the fraction by which each chemical contributes to the ∑TU. Using this approach we could for example estimate how big a fraction of the toxicity of pesticides measured in 19 Danish streams could be attributed to pesticides no longer allowed for use in Denmark, compared to the currently used pesticides (4). Also, the use of the TU-approach has shown that in most cases, even when many pesticides are detected in a water or sediment sample, toxicity is usually driven by relatively few compounds (4, 25).

Do Synergists Play a Role? We stated above that approximately 90% of pesticide mixtures could be described by CA. But this still leaves 10% deviating from the model. Of these, 172 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the mixtures that interact synergistically, thereby inducing an effect that is higher than predicted by CA, are of the largest concern in a risk assessment perspective. In a recent review we evaluated all available mixture studies on pesticides on aquatic organisms to identify, which pesticides might act as synergists (27). A synergistic mixture was defined as a mixture where the observed EC50 was less than two-fold smaller than the EC50 predicted by CA. The study showed that for 95% of the 69 cases where synergy was observed, either azole fungicides or acetylcholinesterase inhibitors were part of the mixture. Azole fungicides are known to inhibit cytochrome P450 enzymes active in phase I metabolism of xenobiotics, and acetylcholinesterase inhibitors, such as organophosphates and carbamates inhibit esterases, which are likewise involved in phase I metabolism. Hence, for pesticides in the aquatic environment it seems that most synergies involve interactions on xenobiotic metabolism. The size of the synergy was rarely above 10-fold. Extrapolating the time of the experiments where synergy could be observed beyond the usual 48 hours used for many aquatic tests could, however, increase synergy ratios to 40-60-fold decrease in EC50 compared to mixtures without the synergist (33). To get an insight in the probability of synergy occurring under more natural conditions than those obtained in laboratory studies, experiments were conducted in mesocosms, where the sorption and degradation of the pesticides could occur under more natural conditions and effects on aquatic communities could be studied. The pyrethroid esfenvalerate was added to the mesocosms in concentrations corresponding to 5, 10 and 25% spray drift events on a 30 cm water column, and the azole fungicide prochloraz was added at a concentration of 90 µg/L, corresponding to a severe run-off event (34). An 8 to 14- fold enhancement of the esfevalerate toxicity was observed for a range of pelagic macroinvertebrate species during the four weeks of observation, while others were unaffected or even increased in population size due to the decreased competition from more sensitive species (34, 35). The study was criticized for using too high concentrations of the synergists, as the µg/L concentrations used are far from the base-flow concentrations usually being in the ng/L range (See Table 1). During the next years, we therefore investigated how low the concentrations of synergists, such as the azole fungicides prochloraz, propiconazole and epoxiconazole, should be to induce a significant synergistic effect (>two- fold). We did so in different test setups using different species and endpoints (24, 36, 37). The conclusion was that the lower threshold for synergy was in the range of 6.4±0.8, 58±7.5 and 40±15 µg/L, for the three azoles, respectively, which is above the base-flow concentration measured and may be achieved only during severe storm-flows (17). How low concentrations that can induce synergy within the acetylcholinesterase inhibitors are less well investigated. Studies on salmon and acetylcholinesterase activity in their brain tissue, however, showed that diazinon and chlorpyriphos were synergistic when combined at 7.3 and 0.1µg/L (38). These results were related to measured aquatic concentrations of diazonon and chlorpyriphos of 6.0 and 0.5 µg/L (38). Hence, it seems that organophosphate insecticides can act as synergists at environmentally realistic concentrations, even though the reported concentrations in the µg/L range is also in the high end of more frequently occurring concentration ranges. 173 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Recently, with the new regulation of Plant Protection compounds in Europe, potential effects of adjuvants and known synergists have come into focus (32). The known pesticide synergist piperonyl butoxide (PBO) have received attention in terms of its potential to induce synergy when occurring at environmentally realistic concentrations (39, 40). So far, however, no severe synergistic interactions have been observed. In the study by Giddings et al (2016), where synergy between PBO and pyrethrins in the sensitive amphipod Hyallella azteca was systematically investigated, synergy was found at PBO concentrations > 4µg/L. The size of the synergistic interactions, however, never exceeded two fold (39). Hence, in a regulatory perspective the synergy was very small. The lack of increase in effect of formulation products under aquatic conditions was also found for ten herbicides representing seven modes of action being tested as both formulated and technical compounds on Lemna minor and algae (41). The only herbicide where larger toxicity was found for the formulated herbicide compared to the technical was for glyphosate, where the glyphosate formulation containing polyethoxylated tallow amine POEA was used. POEA is known to be toxic in itself (42, 43), hence, it is no surprise that the mixture had a higher toxicity than the technical compound. The reason that none of the other formulation compounds increased the herbicide effect is most likely that they are primarily surfactants and penetration oils that need to be present in high concentrations in the spray droplet to enhance herbicide uptake. When diluted in the aquatic environment their uptake enhancing effect disappears. For synergists as PBO, which interact with the detoxification enzymes inside the organisms in a similar way as the azole fungicides, the lower threshold for synergy will occur at a concentration where the fraction of enzymes being affected is too small to play a significant effect for pesticide detoxification. Hence, it can be concluded that even though synergies are interesting from a scientific point of view and may be of importance in certain cases, from a risk assessment perspective, including all pesticides present in surface waters in a cumulative risk assessment is of greater importance if the aim is to catch the full toxic potential of pesticides in a water sample.

Low Dose Effects of Herbicides Aquatic plants and algae are the groups of organisms most sensitive to herbicides. A literature study on herbicidal activity towards aquatic algae including > 120 herbicides, showed no specific herbicidal mode of action to be particularly toxic to aquatic plants and algae (41). In reality, however, combining occurrence and toxicity, photosystem inhibitors are among the herbicides most often found to contribute to effects on submerged plants and algae. In North America, atrazine has been a big issue (44), while European studies on environmental samples, applying the toxic unit approach to identify which herbicides contribute most to the joint toxicity towards algae, also find PSII inhibitors such as isoproturon, diuron, linuron, simazine and terbuthylazine to top the list (4, 45). The question is, whether the concentrations monitored have an effect on the aquatic community. 174 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


To study pesticide effects on aquatic algae and macrophyte communities, three basic approaches are available: creating species sensitivity distributions (SSD’s) based on laboratory derived EC-values for a range of species, conducting microcosm studies or analyzing field data trying to isolate the impact of pesticides on populations (46). Using the SSD-approach, the sensitivity of a range of species is tested, and under the assumption of the tested species being representative of all species, and that their sensitivity distribution is log- normally distributed, the aquatic concentration protecting 95% of the species (The five percent hazard concentration: HC5) can be calculated (Figure 2). In a study on ten species of aquatic macrophytes and an epiphyte community on terbuthylazine and metsulfuron- methyl, we found the HC5 (based on EC50) to be 11 and 39 µg/L for terbuthylazine and 0.031 and 0.014 µg/L metsulfuron-methyl at two different irradiance regimes (47). For terbulhylazine the HC5 values range in the high end compared to SSD’s for another five photosystem II inhibitors of 1.8-10 µg/L (48), while the metsulfuron-methyl HC5 is similar to the ALS-inhibitor HC5 of 0.018 µg/L presented in Giddings et al. (2014) (49). SSD’s do, however, not take species interactions into account, as do microcosm experiments.

Figure 2. The figure shows an example of a Species Sensitivity Distribution (SSD) for aquatic macrophytes. The sensitivity of the species, here given as EC50, is ranked and the distribution is described by a log-logistic curve. The 5% Hazard Concentration (HC5) describes the concentration below which 95% of the species are below their EC50. The data are from Cedergreen et al. (47)). 175 Duke et al.; Pesticide Dose: Effects on the Environment and Target and Non-Target Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


A larger study comparing the SSD-approach with microcosm studies for nine herbicides largely concludes that HC5 values can be used to set benchmark concentrations for environmental effects based on results from microcosm studies (48). To see if this is also the case for mixtures, Knauer and Homme (2013) tested a mixture of three PSII inhibiting herbicides in microcosms at concentrations jointly summing up to HC5 and HC30 for algae SSD’s. They found no measurable effects on algae and macrophyte growth or photosynthetic activity in the HC5 treatments, while significant effects were found in the HC30 treatments (50). They therefore conclude that HC-values in combination with mixture models can be used to derive benchmark concentrations protecting the aquatic algae and macrophyte community. Using the European benchmark concentration for sums of toxic units (∑TU) for algae of 0.1 (32) and applying it to pesticide monitoring data from 19 Danish streams (10 streams with high agricultural pressure), showed no exceedance for the 19 base flow measurements, while 81% of the measurements conducted in the nine agricultural stream during storm-flow exceeded the benchmark of 0.1 (n= 37). Results from four agricultural streams in Sweden during nine years also showed that the low baseflow concentrations only rarely exceeded the benchmark for algae (

Low Dose Effects of Pesticides in the Aquatic Environment - ACS

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