Environmental Fate and Safety Management of Agrochemicals

Chapter 11

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Concentrations of Herbicides Used in Rice Paddy Fields in River Water and Impact on Algal Production S. Ishihara, T. Horio, Y. Kobara, S. Endo, K. Ohtsu, M. Ishizaka, Y. Ishii, and M. Ueji National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan

Plankton and periphyton algae communities are important for maintaining the proper function of aquatic ecosystems. The purpose of this study is to investigate exposure characteristics of rice herbicides in Japanese river and the effects of these herbicides to fresh water micro algae. Distribution and variation of 16 rice herbicides in the Sakura River and the Lake Kasumigaura were monitored during rice growing periods for two years (2001-2002). The highest concentrations were observed at midstream of Sakura R. and these levels were from 0.12 to 8.8 ppb in the middle of May. They were diluted about 20 to 50 times at the center of L. Kasumigaura. The sensitivities of the 4 unicellular algal species to 14 rice herbicides were compared, by using 72-h EC50 (50% growth inhibition concentration at 72-h after treatment) values. The order of relative sensitivity to these herbicides were Selenastrum capricornutum > Merismopedia tenuissima ≥ Achnanthes minutissima ≥ Chlorella vulgaris. Further, to find the sensitivity range of native species, the effects of 6 rice herbicides (Bensulfuronmethyl, Imazosulfuron, Simetryn, Dimethametryn, Pretilachlor and Cafenstrole) to the freshwater algal species isolated from the natural environment such as

112

© 2005 American Chemical Society Clark and Ohkawa; Environmental Fate and Safety Management of Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

113


rice paddy and streams were investigated. As a result, some Senedesmus strains indicated low sensitivity to the pretilachlor and cafenstrole. In addition, some pennate diatoms and Senedesmus strains indicated low sensitivity to the triazine herbicide.

Plant communities are important for maintaining the proper function of freshwater, estuarine and marine ecosystems. Algae associated with the plankton and periphyton form the base of most food chains, produce oxygen, and play important role in cycling of nutrients (1). In Japan, more than half of agricultural land is paddy fields. The rice herbicides are easy to run off especially because those are applied directly to the surface water of the paddy field. Thus, some rice herbicides are detected frequently in river water of Japan at the low ppb levels for some months after the rice-planting period (2-6). Alterations of a phytoplankton community as a result of toxic stress may affect the structure and functioning of the whole ecosystem (7). However, there have been few reports on the influence of thericeherbicides to phytoplankton (8-11). Therefore it is important to assess the effects of these herbicides on primary production in riverine, lake and marine ecosystems. Since 2000, MAFF (Ministry of Agriculture, Forestry and Fisheries) Japan has been required that registrants submit phytotoxicity data (Algal, Growth Inhibition Test) for CPPs (Crop Protection Products) registration. This test results show only the adverse effect on unicellular green algae, and cannot estimate the adverse effect on a phytoplankton community by CPPs. One of the key issues that need to be resolved in toxicity testing is the great variability in sensitivity among species (12-13). Because of geographical condition, Japanese rivers are steep and its flow is very fast. Residence time of thefloatingspeases at certain point in a river seems much shorter than the life span of planktonic algae. Therefore, the periphytonic algae, mainly diatoms are important organisms in aquatic ecosystem in Japan. Also, it is important to select appropriate species in order to evaluate the proper function of native environment. However, the data on phytotoxicity testing are limited, which makes realistic and scientifically sound risk characterizations difficult. The objectives of this study are to investigate exposure characteristics of rice herbicides in Japanese river, to compare the relative sensitivity of various algal taxa to herbicides, and to conduct more realistic and scientifically sound risk characterizations.

Clark and Ohkawa; Environmental Fate and Safety Management of Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

114

Materials and Methods


Herbicide concentrations in the river The location of monitoring stations is shown in Figure 1. Lake Kasumigaura is the second largest lake in Japan, and located on the 60 km northeast of Tokyo. Sakura River is one of the mainriverswhich flow into L. Kasumigaura and many paddy fields are situated in the river basin. Herbicides concentrations were monitored at 7 locations in 2001. St. 1 is located at mountainside of Mt. Tsukuba and one of the headstream of Saka R, which is a tributary of sakura R. There are neither paddy fields nor houses above this point. St. 2 is located at down stream of Saka R., which frow trough extensive paddy field area. St. 3 is located at the midstream of Sakura R. and many paddy fields are also situated around the riverside. St. 4 is located on the downstream of Sakura R. This station is located at the center of a Tsuchiura City. St. 5 is located at the river mouth of the Sakura R. St. 6 is located in Tsuchiurairi Bay. St. 7 is located in the center of the L. Kasumigaura.

Figure 1. The location of monitoring station Water sampling period was from March 20 to September 19 in 2001 and St. 2, St. 3 and St. 4 were investigated from April 22 to August 19 in 2002 again. Twelve rice herbcides (cafenstrole, dimepiperate, dimethametryn esprocarb, mefenacet, molinate, pentoxazone, pretilachlor, pyributicarb, pyriminobacmethyl, simetryn and thiobencarb) were analyzed by gas chromatography (GC/FTD: SIMAZU GC-17A), and 4 rice herbcides (bensulfuronmethyl, daimuron, imazosulfuron and pyrazosulfuronethyl) were analysed by Liquid Chromatograph/Mass Spectrometer/Mass Spectrometer (LC-ESI-MS/MS :



115 API3000LC/MS/MS System (Applied Biosystems/MDS SCIEX)). For GC analysis, each of water sample was filtered through a glass filter (Whatman GF/F 0.7μπι) and its pH was ajusted to be 6.5. One litter of filtrate was passed through solid-phase extraction cartrige (Waters Sep-Pak tC18) and eluted with dichloromethane. The eluate was concentrated in vacuo and dissolved in 2ml acetone. For LC-ESI-MS/MS analysis, each of water sample wasfilteredthrough a glass filter (Whatman GF/F 0.7μπι) and kept frozen until the herbicides analysis. Equal amount of internal standard substance contain acetonitrile was added to the sample and centrifuged for lOminutes at 20,600*g and then 5μ1 of clear supernatant liquid was injected to LC-ESI-MS/MS.

Toxicity tests on standard species Algae Tested 4 algae were axenic unicellular freshwater algae, Selenastrum capricornutum Printz ATCC 22662 (Clorophyceae), Chlorella vulgaris Beijerinck NIES-227 (Clorophyceae), Achnanthes minutissima Kuetzing NIES71 (Bacillariophyceae) and Merismopedia tenuissima Lemmermann NIES-230 (Cyanophyceae). S. capricornutum was obtained from the American Type Culture Collection, USA. The other three strains were obtained from the Microbial Culture Collection in the NIES (National Institute for Environmental Studies), Japan. S. capricornutum and C. vulgaris, have been recommended as standard strains for the OECD algal toxicity test (14). Each strain was maintained on Medium Csi (pH7.5) (15) 1.5% agar slants.

Herbicides The 14 herbicides tested are listed in Table 1, with their chemical properties, amounts of shipment (2000), typical aplication rates (g/lOa) and the registration year in Japan. All herbicides used were analytical grade.

Algal assay The algal inoculum was taken from preculture in the exponential growth phase. Preculture and assay were conducted at 2 3 ± 2 ° C under continuous illumination of approximately 3,0001ux using a shaking incubator at lOOrpm (SIBATA, RS-200). The test algae were cultivated in 300ml sterile foamplugged Erlenmeyer flasks, each containing 100ml of Medium C (pH7.5) for S.


116 capricornutum C. vulgaris and M tenuissima, 100ml of Medium Csi (pH7.5) for A. minutissim. The initial algal cell concentration in the test culture was approximately Ι* 10 cells/ml for S. capricornutum and C. vulgaris, 3xl0 cells/ml for M. tenuissima, 5x10 cells/ml for A. minutissim. The concentration range in which effects were likely to occur was determined from range-finding tests. For the definitive test (EC value finding tests), five concentrations arranged in a geometric series in a ratio of 1.6 to 2.5 depending on test conditions. However, definitive testing was not performed i f the highest herbicide concentration tested (water saturation concentration or l,000mg/L) results in less than a 50% reduction in growth. DMSO (dimethyl sulfoxide) was used as the vehicle to dissolve herbicides at a concentration of 0.1%(maximum). Algal assays were performed by three replicates at each concentration. There were also three replicates of the controls that contained 0.1% DMSO without test herbicides. Every test species cell counts was performed after 0,24,48 and 72 h using a PAS-FCM (Particle Analysing System-Flow Cytometer, partec GmbH). For the E C value finding tests, herbicides concentrations were measured at the beginning and the end of the assay using a GC/FTD or HPLC/UV. 4

5

3

50

Table I. Testing herbicides and their mode of action, chemical family, common name, chemical properties, amount of shipment (2000), typically applied quantities (g/lOa) and registration year in Japan No.

Common name

Thiobencarb 2

Esprocarb

3

Mollnate

4

Dimepiperate

5

Pretilachlor

6

Mefenacet

7

Cafenstrole

8

Dimethametryn

9

Slmetryn

Mode of action Inhibition o f lipid synthesis

Water Chemical family solubility (mg/l)

Tiocarbamates

··

-

«

Inhibition o f Veiy Long Chain Fatty Chloroacetarnides Acids (Inhibition o f cell division)

• Inhibition of photosynthesis at

-

470

1969

310

1988

900

2.9

240

. 260

1971

20

4.0

300

75

1986

40

280

1984

3.2

no

470

1986

Others

2.5

3.2

70

no

1996

Triazincs

50

3.8

6

22

1975

Triazmes

400

2.6

45

77

1969

-0.5

330

230

1975

II

Quinones

Daimuron

150 150

4.1

Bentazone

14

3.4 4.6

4

Qulnoclamine (ACN)

Imazosulfuron

30 4.9

50

10

13

570 22

1.5

270

86

1968

17

63

1987

9

14

1993

150

550

1974

Inhibition o f acetolactate synthase

Sulfonylureas

120

0.62

»

Sulfonylureas

310

0.05

Unknown

Common rcgistralcd Shipment Registration application year in 2000 ratea ( a X . t o r k l ) In Japan (g/10a)

Oxyacetamides

Benzothiadiazinones

12 Bensulfuronmethyl

11


50

Ureas

1.2

2.7


117 Toxicity test on native species


Algae Unialgal clonal strains (pennate diatoms and green alga, Senedesmus sp.) were isolated from natural population samples collected at the paddy field, the drainage canal (near the St. 2, see Figure 1) and the river (St. 1, 3 and 4) in August or October, 2002. Each of strains was maintained on Medium Csi (pH7.5) 1.5% agar slants. Total number of isolated strains were 42(pennate diatoms) and 20 (Senedesmus sp.)

Herbicides The 6 rice herbicides (Bensulfuronmethyl, Imazosulfuron, Simetryn, Dimethametryn, Pretilachlor and Cafenstrole) were used for the algal assay.

Algal assay The algal inoculum was taken from preculture in the exponential growth phase. Preculture and the assay were conducted at 23±2°C under continuous illumination of approximately 3,0001ux. The test algae were cultivated in 96-well microplate, each containing 0.2ml of Medium Csi (pH7.5). The initial algal cell concentration in the test culture was approximately 5*10 cells/ml for Senedesmus sp. and 3xl0 cells/ml for pennate diatoms. For the E C value finding tests, ten concentrations arranged in a geometric series in a ratio of 2.0. Cell density was measured after 96 h using a spectrophotometer (BIO-RAD, Model 550). The absorbance values of the sample was measured at 680nm and 96 h EC o value was calculated. 3

3

50

5

Results and Discussion Herbicides concentrations At the St. 1, any herbicides were not detected through the period. At the St. 2, fourteen kind of herbicides were detected. The herbicides included cafenstrole, dimepiperate, dimethametryn, esprocarb, mefenacet, pentoxazone, pretilachlor, pyributicarb, pyriminobac-methyl, simetryn, bensulfuronmethyl, daimuron, imazosulfuron and pyrazosulfuronethyl. The herbicides detection period of this



118 station was about one month. Summed concentrations of detected herbicides in water samples of Sakura R. and L. Kasumigaura is shown in Figure 2. Total of 16 herbicides (The above-mentioned herbicide plus molinate and thiobencarb) were detected at Sakura R. The highest concentrations were observed at midstream of Sakura R. (St. 3) and these levels ranged from 0.12 ppb for pyriminobac-methyl on May 8, 2002 to 8.8 ppb for daimuron on May 14, 2002. ppb in the middle of May. The concentrations of detected herbicides were approximately the same level in the midstream (St. 3) and downstream (St. 4) of Sakura R. They were diluted about 2 times at the river mouth, about 10 times at Tsuchiurairi Bay, and about 20-50 times at the center of L. Kasumigaura. For most rice herbicides detected in Sakura R. and L. Kasumigaura, the maximum detected concentration levels were below low ppb lebel. Recently, the concentration level of individual herbicide detected in surface water tend to decrease in Japan (3,4,16). Especially during 90's, this tendency become obvious. This phenomenon is probably the results of reduction of rice cultivation areas and increase in number of active ingredient in the market. Also, it was probably affected by the development/application of low-dose, high-potency herbicides such as sulfonylurea herbicides has advanced.

Herbicide susceptibility in four unicellular freshwater algae Effects of 14 kinds of rice herbicides to 4 kinds of algae were investigated, and 39 E C values were determined (12; S. capricornutum, 10; A. minutissim, 9; M. tenuissima, 8; C. vulgaris ). S. capricornutum is highly sensitive to tested herbicides and it is the most popular test algal strain for toxicity tests. Also, S. capricornutum have been recommended as standard strains for the OECD algal toxicity test. On the other hand, C. vulgaris is insensitive to tested herbicides. Although the trend of C. vulgaris E C is very different from the case of S. capricornutum. C. vulgaris have still been recommended as standard strains for the OECD algal toxicity test. A. minutissim is one of periphytonic pinnate diatoms which commonly observed in Japanese rivers. Similar to C. vulgaris, it has low sensitivity on most of herbicides as compared with S. capricornutum. Especially, sensitivity to sulfonylurea herbicides is very low. M. tenuissima is a kind of blue-green algae and is the same order of algae with Genus microcystis which causes blue-green algae waterbloom. Usually blue-green algal speces is difficult to do the subculturing on solid medium however this M. tenuissima has an advantage of performing subculturing easily on solid medium and it is highly applicable for toxicity tests. Similar to algae explained above such as C. vulgaris and A. minutissim, M. tenuissim has also low sensitivity on most of herbicides as compared with S. capricornutum. Though, it has very high sensitivity to 50

50



119

ο •f-H

ο

α ο U

(ppb)

Mar. Apr,

Figure 2. Herbicide concentrations in water samples of Saka R, Sakura R. (2001, 2002) andL. Kasumigaura (2001)



120 sulfonylurea herbicides. The order of relative sensitivity was S. capricornutum > M. tenuissima^ A. minutissim^ C. vulgaris (Figure 3). Triazine herbicides and quinoclamine, having the mode of action in inhibition of PS II (Photosynthesis at photosystem II) had low variability on sensitivities in different algal taxa. On die other hand, Amide herbicide such as Pretilachlor and Cafenstrole as well as sulfonylurea herbicides of bensulfuronmethyl and imazosulfuron had great variability on sensitivities in different algal taxa. These herbicide have in other the mode of action in inhibition of cell division or in inhibition of acetolactate synthase rather than in inhibition of PS II. Carbamate herbicide showed relatively low toxicity on algae. Daimuron and bentazone exhibit low toxicity on all the tested species (Figure 3).

>

C. vulgaris

>

A. minutissima

M. tenuissima

0.1 I Φ:

ECso

OD Q0B>C3>O Χ

OO

S. capricornutum

> •

>

> Ο

O m

K) DO >

X

10

100

EC50

(pP^> 72-hi*)

1000

10000

/ S : Inhibition of lipid synthesis ^ (dimepiperate, esprocarb, thiobencarb, molinate) h> : Inhibition of photosynthesis at photosystem II -K : Others (dimethametryn, simetryn, quinoclamine) (Daimuron, Bentazone)

0

:

Inhibition of VLCFAs (cafenstrole, mefenacet, pretilachlor)

I I : Inhibition of acetolactate synthase (ALS) (bensulfuronmethyl, imazosulfuron)

Mode of action

Figure 3. EC values of four unicellularfreshwateralgae at fourteen rice herbicides 50

Herbicide susceptibility in freshwater pennate diatoms and green alga, Senedesmus sp. Figure 4 shows the E C values of 6 herbicides for individual pennate diatoms and Senedesmus sp. isolatedfromnatural population strains. 50


121 JQQ QQ0T G r C e n cllgcl6 (Senedesmus sp. & Selenastrum capricornutum)

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ιιιι

§„ο

'un 6 Πι! us I n n i -i ! _il

I Δ ι I

1

ills . ί 2 ϊ IS (sampling point)

ft

ο* (Herbicides)

ioo,ooorP

ennate

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•••••

ο o

ooooo

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ι ο,οοο f 1,000 ω

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IIIII Ktit îîIII ιιιιι ÎÏÎÎÎ it A -«-- 3 11 a' -ÎI- -ft— 1 4(£L . w£Ο Κ 35

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M. tenuissima^A. minutissim è C. vulgaris. Six rice herbicides to the freshwater algal native species. As a result, some strains indicated low sensitivity to rice herbicides. However, these herbicides are unlikely to cause acute adverse impacts on primary productivity of the Sakura R. It is difficult to evaluate the realistic and scientifically sound risk characterizations on the primary production in aquatic system of herbicides using only a result of the toxicity test to unicellular green alga. Therefore, we should take into account the species/strain differences in the susceptibility when the aquatic plant risk was assessed. Furthermore, to establish phytotoxicity assessment scheme (including level of progression for phytotoxicity testing), we should decide test species/methods of aquatic plants (including algae) at an early date.


123

Acknowledgements


We would like to express our sincere thanks to a number of colleagues at Tokyo University of Agriculture and Technology, National Agricultural Research Organization, Japan and Agricultural Chemicals Inspection Station, Japan for supporting this research.

References 1.

Gary M . R.; Fundamentals of aquatic toxicology Second edition. Taylor & Francis. London, U K (1995) 2. Shiraishi H., Pura F., Otsuki A. and Iwakuma T.; The science of the Total Environment. 1988, 72, 29-42. 3. Maru S.; Special bulletin of the chiba prefectural agricultural experiment station. 18, Chiba-shi, Japan, 1991. 4. Nakamura K.; Bulletin of the saitama agricultural experiment station. 46, Kumagaya, Japan, 1992. 5. Nohara S. and Iwakuma T.; Chemosphere. 1996, 33, 1417-1424. 6. Okamoto Y., Fisher R.L., Armbrust K.L. and Peter C.J.; Journal of pesticide science. 1998, 23, 235-240. 7. Mosser. J.L., N.S. Fisher and C.F. Wurser.; Science. 1972, 176, 533-535. 8. Hatakeyama S., Fukushima S., Kasai F. and Shiraishi H.; The Japanese Journal of Limnology. 1992, 53, 327-340. 9. Kasai F., Takamura N. and Hatakeyama S.; Environmental Pollution. 1993, 79, 77-83. 10. Hatakeyama S., Fukushima S., Kasai F. and Shiraishi H.; Ecotoxicology. 1994, 3, 143-156. 11. Nishimoto H.; Research Bulletin of the Aichi ken Agricultural Research Center. 1998, 30, 71-77. 12. Roshon R, McCann JH, Thompson DG, Stephenson GR.; Canadian journal of forest research. 1999, 29, 1158-1169. 13. Susan A. F.; Impacts of low-dose, high-potency herbicides on nontarget and unintended plant species. SETAC PRESS. Pensacola, FL. (2001) 14. OECD; OECD Guidelines for Testing of Chemicals, Alga, growth inhibition test. Organization for Economic Co-operation and Development, Paris. (1984) 15. GEF; GEF List of Strains '97, Microalgae and Protozoa. Global Environmental Forum, Tokyo, Japan. (1997) 16. NIES, Japan; Report of special research from the National Institute for Environmental Studies, Japan. SR-29-'99. (1999)


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