Chlorinated Paraffins and the Environment. 2. Aquatic and Avian Toxicology John R. Madeley” and Robin D. N. Birtley Imperial Chemical Industries Limited, Research and Development Department, Mond Division, The Heath, Runcorn, Cheshire, P.O. Box No. 8, WA7 400, United Kingdom
The toxicity of certain chlorinated paraffins (CPs) to fish and birds is presented. No toxic response could be demonstrated. A high molecular weight CP (42%C1 w/w) containing a I4C radiolabel was taken up into the tissues of rainbow trout and mussels. Comparison of CP concentration in tissues, as measured by a thin-layer chromatographic technique and 14C, indicated that mussels expelled the CP as the parent compound. Trout, however, appeared to metabolize the material. The ability of aerobic microorganisms to oxidize a range of CPs depends upon their previous acclimatization, the hydrocarbon chain length, and its degree of chlorination. Short-chain compounds are the most readily oxidized a t chlorine levels below 60% (w/w). Complete breakdown has been demonstrated for a long-chain CP over a period of 8 weeks. Anaerobic microorganisms, under the experimental conditions examined, did not degrade a specific CP in 30 days.
I. Introduction A sensitive analytical method has been developed for the measurement of chlorinated paraffins (CP) in a range of environmental samples (I).Use of this method ( 2 )has enabled some knowledge to be gained of the distribution of these compounds in the aquatic and terrestrial environments, birds, and man. It appears that CPs are widely distributed, albeit at low concentrations; therefore, it becomes important to know of their toxicity, behavior, persistence, and fate in relation to these levels. The aim of this particular paper in the three-part series is to present results of laboratory studies on toxicity, bioaccumulation, and biodegradation in the aquatic environment, with some information on avian toxicity. Mammalian toxicology, although summarized here, is presented in detail elsewhere ( 3 ) .
2. E f f e c t s o n Aquatic Organisms 2.1. Introduction. Laboratory studies to determine the toxicity, bioaccumulation, and possible biomagnification of CPs have, in the past, been hampered by the lack of a sensitive analytical method. With the benefit of such a method, our own investigations have been designed to highlight areas where environmental problems could possibly arise and indicate where further assessments may be required for a more complete understanding of CP behavior. 2.2. Acute Toxicity and Sublethal Observations. Acute toxicity studies have been carried out to ascertain the 96-h LC50 value-the concentration required to kill 50%of fish in 96 h-for Cereclor 42 (C20-30 42%C1 w/w) by using rainbow trout (Salmo gairdneri) as test species. Cereclor is a trade mark of Imperial Chemical Industries Limited for chlorinated paraffins. Concentrated emulsions of the C P were prepared and subsequently diluted for exposure of 10 fish per concentration. Static bibassays were carried out a t 15 f 1 OC, emulsions being changed every 24 h. Emulsion concentration was determined by infrared spectrophotometry after extraction of CP in carbon tetrachloride. Some loss of CP from emulsion occurred, particularly from the higher concentrations; quoted values are means from several sets of analytical figures. 0013-936X/80/0914-1215$01.00/0 @ 1980 American Chemical Society
Over 96 h CP emulsions were nontoxic to rainbow trout at a mean concentration of 770 ppm (range 520-1630 ppm). No sublethal effects were observed owing to the opacity of emulsions. No unusual fish behavior was noted. The CP can therefore be considered as nontoxic over a short exposure period. By comparison, a PCB, Aroclor 1242, was found to be toxic to rainbow trout; the 96-h LC50 value was between 49 and 77 ppm. Similar results for a series of CPs were obtained by Johnson ( 4 ) who demonstrated 96-h LC50 values in excess of 300 ppm to rainbow trout and bluegills (Lepomis macrochirus). However, at concentrations of 40 ppb, for an unidentified CP, he demonstrated certain neurotoxic effects in flowthrough experiments. Loss of motor function and immobilization were described. These observations have been confirmed by Svanberg et al. ( 5 )over exposure periods in excess of 14 days. Bleaks (Albernus albernus), exposed to 1.0 and 0.1 ppm of Huls chlorparaffin 70C (C10-13 70% C1 w/w) added to the test water from acetone solution, showed signs of disorientation and tetanic spasm which led to the death of three individuals. Short-term toxicity tests (96 h) demonstrate the very low acute toxicity of CPs to the test species examined. Neurotoxic symptoms, observed during more prolonged exposure, appear to be restricted to the lower molecular weight compounds. The effects could be related to their higher water solubility, possibly enhanced by method of addition, as well as the higher “biological activity” of these CP grades. 2.3. Bioaccumulation in the Laboratory. It has been suggested ( 6 ) that the high molecular weight of CPs would appear to limit their potential for bioaccumulation in the aquatic environment. In addition, their very strong tendency to adsorb on suspended particles would reduce the availability of CPs to food-chain organisms. This suggestion was based in part on the work of Zitko (7) and Lombard0 et al. (8);test fish fed on CP-dosed food showed that the compounds were detected but were not accumulated to any significant extent in tissues. Since then, however, Svanberg et al. (5) have detected an accumulation of organic chlorine in fish exposed to low concentrations of a low molecular weight CP in water. In the studies described here, CP was uniformly mixed with food to examine specifically this route of administration to the common mussel ( M y t i l u s edulis) and rainbow trout (Salmogairdneri). Experiments were carried out by using a C P based on a wax feedstock (C20-30)chlorinated to 42%by weight (Cereclor 42). The availability of I4C centrally labeled pentacosane (C25),subsequently chlorinated to 42%by weight, allowed concentrations of CP to be followed in food and animal tissues by radiocarbon analysis. For experimental purposes the chlorinated pentacosane was mixed with Cereclor 42 before use. In the case of mussels, suspended yeast cells were finally chosen as the CP-dosed food source; attempts to contaminate marine unicellular algae uniformly had previously proved unsuccessful. Commercial trout diets (Cooper Nutrition No. 4 pellets) were similarly dosed with CP as the sole food for rainbow trout. All test organisms were maintained in glass-reinforced plastic tanks; dosing lines were constructed from glass. Test solutions were maintained at 16 f 2 “C for mussels and 12 f 3 OC for rainbow trout. Volume 14, Number 10, October 1980 1215
The test and control populations were sampled regularly during the feeding of dosed food, and also after clean diet substitution. Six mussels and three fish were sacrificed on each sampling occasion and dissected into appropriate tissue and organs for 14C examination. Analysis was carried out by combustion in a Packard sample oxidizer (Model 306) where appropriate, followed by counting in a Packard Tricarb scintillation spectrophotometer (Model 3320). 2.3.1. Rainbow Trout Study. Separate rainbow trout populations each of 40 individuals were fed diets containing average concentrations of 47 ppm (w/w dry) and 385 ppm (w/w dry) CP for a period of 35 days; a similar control group was fed the same but uncontaminated diet. Subsequently all fish were fed control diets for a further 49 days. Table I compares the 14Cconcentration in tissues of fish on the final days of feeding both the dosed and clean diets. Quantities found in tissues after 35 days were closely related to food dose levels, and, further, the 14C,remaining after depuration was related to the amount originally accumulated in the tissues. The uptake and loss of 14Cin the tissues of fish fed on diets containing 47 ppm C P are illustrated in Figure 1. During accumulation a wide variation of 14C in gut tissue was determined; this however, could be expected owing to difficulties in dissection and bearing in mind the administration route.
By comparison the uptake of 14C in liver was much more uniform. The concentration in both liver and gut approached that administered in the food. Other tissues showed less uptake, so that the calculated total body level was well below the concentration administered and reached a plateau by day 35. Liver and gut samples lost 14Crapidly and markedly within 49 days, a t a rate much greater than other tissues. The form of all depuration curves indicated that I4C in all tissues would reach low residual levels within a period in excess of 50 days. Fish populations were carefully scrutinized for any unusual symptoms throughout the test. One death occurred in the control tank. The condition of individuals was estimated by measurement of the “condition factor” (9);these were always >1, indicating their prime condition throughout the test. 2.3.2. Mussel Study. Dry baker’s yeast was dosed with CP a t a mean concentration of 524 ppm (w/w dry) before suspension in freshwater for addition to the test tanks. These treated yeast cells were fed to mussels for 47 days; controls received untreated yeast. After this time all mussels received untreated yeast for a further 56 days. The uptake of 14Cby mussels continued up to the end of the 47-day accumulation period (Table 11). Compared to the concentration in food this uptake was small. Within the
Table 1. 14C Concentration in Fish Tissues Measured on the Final Days of Feeding CP-Containing Diets and Clean Diets treatment
exposure period, days
liver
diet containing 47 ppm (dry wt) subsequently fed clean diet diet containing 385 ppm (dry wt) subsequently fed clean diet
35 49 35 49
29.5 2.8 263 21.8
a
mean concn I 4 C in tissues (ppm dry wt) gut flesh remains
36.4 4.8 353 47.3
2.5 1.4 23.1 17.2
6.8 3.1 66.3 29.8
totala
10.3 3.3 100.6 31.6
Total derived from measurements of tissue weights and 14C concentration for individual fish.
I
ACCUMULATION
““1
., 0
0
I
X
I
DEPURATION
X
0
10
20
i0
40
50
70
60 TIME
80
(DAYS)
Figure 1. Uptake and loss of I4C by rainbow trout (diet containing 47 mg of Cereclor 42/kg): (0) liver: (X) gut: (0)flesh; (A)total body (calculated). Regression curves derived from equations: (i) accumulation phase, c = k l log e(t 1); (ii)depuration phase, c = l / [ k * ks(t to)];where c = concentration at time t from start of test, to = start time for depuration phase, k , , k2,and k3 = constants derived by regression analysis.
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Environmental Science & Technology
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mussels the 1% was concentrated in the digestive gland. In all other tissues examined the levels remained below 10 ppm (wlw dry). Mortality was assessed by observation of excessive shell opening; any “gaping” individuals were considered “dead” if, upon removal from the tank, they were incapable of fully closing within 1 min. On this basis deaths recorded were 10 individuals in the test tank and 7 in the control. 2.3.3. Analysis of CP in Fish and Mussels. The experiments undertaken were primarily designed to assess the extent of accumulation, distribution, and loss of CPs from organisms by radiolabel techniques. However, measurement of the parent compound concentration in the same tissues for comparison is capable of providing evidence on the occurrence of CP breakdown. During the course of the 14C studies, certain individuals were retained for analysis by the TLC procedure
figures obtained on the same extracts after TLC separation and quantification. The concentrations of 14C and CP in the extracts were generally similar but very much lower than the 14Clevel obtained after tissue combustion. If one takes into account any losses of CP during preparative stages, these differences would appear significant, leading to the conclusion that not all 14C in fish tissue was present as parent CP. This suggests that some CP was dehydrochlorinated or metabolized to smaller molecules (below Clo) and incorporated into other degradative or synthetic pathways within the animal. The behavior of I4C originally entering the animal from food dosed with CP has been followed through the major steps of extraction and cleanup before TLC separation. Recoveries of 14Chave been compared with those for the same CP dissolved in hexane. Results are presented for two individual fish in Table IV. Recovery of CP from spiked fish tissue was -90% ( I ) . It has also been shown that the recovery of 14C-labeledCP present in hexane was similar, whereas final extracts from experimentally dosed fish contained only 27-33% of 14Coriginally present. Failure to recover major quantities of 14C occurred
( 1 1.
Three individual fish were retained a t the end of the study for this comparative analysis. Table I11 shows the mean 14C values for the tissues examined by direct combustion. Also shown are the mean 14Cvalues on the extracts after cleanup in the CP analytical procedure, together with the mean CP
Table II. Concentration of I4C in Tissues/Organs of Mussels Examined during Accumulation/Depuration (days) tissue/organ examined a
accumulation period (concn of 14C in tissuelorgan, ppm dry weight)
5
day
12
9
19
digestive gland foot gonad gill
remaining tissue total body a
1.8
2.4
4.2
5.1
depuration period (concn Of I 4 C in tissuelorgan, ppm dry weight) 7 21 28 39 49 58
28
33
42
47
155.2 2.6 2.8 3.5 1.aC 4.3
36.6 1.3 1.9 5.3 2.9 5.9
100.7 2.2 3.5 4.4 4.6 6.6
80.5 5.4 1.9 7.0 6.8 11.2
35.1 1.8 1.4 3.3 1.7 5.5
37.0 3.3 2.0 3.1 1.7 2.9
42.2 3.1 1.0 1.9 1.5 2.0
15.8 2.9 2.0 3.6 1.7 2.2
11.3 1.7 1.5 2.5 1.3 3.1
7.0 0.9 0.9 1.5 0.6 1.2
Mean for three individuals. Mean for three separate individuals. One high value (36.0) omitted
Table 111. Concentration of Cereclor 42 in Fish Tissues (Means from Three Individuals) Measured by TLC and 14C Scintillation Counting PPm (dry weight)
Of
I 4 C and Cerecior 42 in tlssues chemical extraction and cleanup, followed by ~
previous feeding regime and sampling date
food containing 385 ppm (w/w
tissues/organs examined
14C concn by combusting and scintillation counting
14C count of extract
CP concentratlon spot intensity measurement
liver
21.8
1.5
6.5
gut
47.3 17.2 29.8
8.7 4.8 5.9
5.4 3.7 5.7
dry)
Cereclor 42, sampled day 49 of depuration period
flesh remains
Table IV. Distribution of 14C in Extracts Obtained during Preparative Stages of the TLC Procedure for Quantification of CP in Tissues major preparative stages in TLC procedure for CP measurement
total I4C in sample extraction ( i ) 1st Soxhlet extraction, 3 h hexane (ii) 2nd Soxhlet extraction, 3 h hexane ( i i i ) residue plus Na2S04after extractions 3. Cleanup: hexane/dimethylformamide (DMF) partitions (i) hexane after DMF extraction (ii) hexane wash (iii) bulked DMF plus 2 % Na2S04after back extraction 4. TLC plate (i) final hexane extract “spotted” on plate 1. 2.
fish A
fish B
14C-iabeied CP in hexane
14c, pg
YO total
14c, c g
% total
14c, pg
% total
62.7a
100a
32.2a
100a
50.3
100.6
43.4
