Chlorothalonil

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jan 1983 to 4 Feb 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted according to "Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians". (ASTM 1980)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Duplicate water samples for chemical analysis of the test substance were taken daily throughout the test from each treatment. The 450 mL samples were siphoned from midway in the water column and placed in 500 mL linear polyethylene bottles and immediately frozen. The samples were then shipped with dry ice to analyze facility for chemical analysis.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

A primary stock solution (821 mg/L) was prepared by adding 0.4105 g of the test substance to a 500 mL volumetric flask and bringing to volume with reagent grade acetone. The other stock solutions were prepared by serial dilution. A seawater and solvent control were run concurrently. The seawater control did not receive any test material. The solvent control received the same volume of acetone added to all exposure aquariums, but no test material.

Test organisms (species):

other aquatic mollusc: Crassostrea virginica

Details on test organisms:

TEST ORGANISM
- Common name: Eastern oyster
- Source: Commercial supplier
- Age at study initiation: < 2 years old
- Weight at study initiation: 1.1 - 4.9 g wet weight without shells (Mean weight: 2.6 ± 1.3 g)
- Length at study initiation: 37 - 65 mm umbo to distal valve edge
- Mean height at study initiation: 53 ± 8 mm
- Feeding: Oysters obtained plankton and other particulate matter from the unfiltered seawater in which they were tested; there was no supplemental feeding.

ACCLIMATION
- Acclimation period: 4 days
- Acclimation conditions: Flow thorugh seawater
- Salinity: 24 - 32‰
- Temperature: 13 - 16°C
- Health during acclimation: There was no mortality during the 48 hours period before the test and oysters appeared to be in good condition
and growing well (≥ 2 mm new shell growth) at the initiation of the test.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

14 - 16°C

pH:

Initiation of the test : 8.2 (in all treatments)
End of the test: 8.0

Dissolved oxygen:

> 100% of saturation (in all treatments)

Salinity:

14 - 27‰

Nominal and measured concentrations:

- Nominal concentration: 1, 2, 4, 8, 16, and 32 µg/L
- Measured concentration: 0.6, 1.6, 3.2, 7.4, 15.2 and 30.8 µg/L, respectively. See Table 1 in "Any other information on materials and methods incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 9 L galss aquaria
- Filled volume: 7 L test solution or control seawater at a depth of 9 cm
- Aeration: No
- Flow rate: 60 L/hour
- No. of organisms per vessel: 10 (randomly distributed to all test chambers after a 2.5 hour equilibration period)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: Approximately 18 mg of tissue/ L of test solution/day or 3.7 g of tissue/L of test solution in the aquaria at anytime

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of test water: Water used to maintain and test the oysters was unfiltered, natural seawater which was pumped from Big Lagoon, a Gulf of Mexico estuary adjacent to the test facility. The pump intake was located about 80 m offshore at a depth of approximately 3 m. Water was pumped through polyvinylchloride (PVC) pipes into an elevated fiberglass reservoir. From the reservoir, water flowed by gravity through PVC pipes into the laboratory. No attempt was made to alter the salinity or temperature of the incoming water.


- Intervals of water quality measurement: Dissolved oxygen concentrations and pH were determined and recorded at 0, 48 and 96 hours in all treatments and both controls in which there were surviving oysters. Salinity and temperature were measured in the solvent control at 0, 24, 48, 72 and 96 hours.


OTHER TEST CONDITIONS
- Lighting: Ambient window light supplemented with fluorescent lighting during daytime working hours


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Before place the oyster into teats container: Oysters were cleaned of attached organisms, ground by hand using a fine-grit grinding wheel to remove approximately 2 - 5 mm of new peripheral shell
- After 96 hours of exposure: The oysters were removed from the test containers and new shell growth of each oyster was measured to the nearest 0.1 mm with a vernier caliper. The mean shell growth of solvent control oysters less the mean shell growth of oysters from each test concentration divided by the solvent control growth and multiplied by 100 gave the percentage reduction in shell growth for oysters from each test concentration relative to the solvent control.


RANGE-FINDING STUDY
- Test concentrations: 2.5, 25 and 250 µg/L

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

7.5 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Percentage reduction of shell growth

Details on results:

An overwiev of the results is provided in Table 1 in 'Any other information on results incl. tables'

Nominal concentrations of > 4.0 µg/L significantly reduced new shell growth of oysters exposed to solvent only. Reduction in shell growth among exposed oysters ranged from 44% in the 4.0 µg/L test concentration to 94% in the 32 µg/L test concentration. Increases in shell growth among exposed oysters of 6 and 7% were measured for oysters exposed to 1 and 2 µg/L, respectively.

There was a significant increase in shell growth of the seawater control oysters of 28%, as compared to the solvent control oysters. It was however not expected that there was any actual solvent effect because the two lowest test concentrations also contained the same amount of acetone and were not statistically different from the seawater control.

Reported statistics and error estimates:

Based on the results of the test, a 96-hour EC50 and 95% confidence limits were calculated. The computer program estimated the EC50 by using the following three statistical methods: moving average angle analysis, probit analysis, and binomial probability. The method selected was determined by the characteristics of the data, that is, the presence or absence of 0% and 100% effect and the number of concentrations in which > 0% <100% effect occurred (Stephan, 1977). For this test, the results of the moving average angle method were used to report the 96-hour EC50.
Shell growth of oysters in all treatments were compared to the shell growth of solvent control oysters by a "Student's" t-test (Steele and Torrie, 1960) to determine if the presence of the test substance significantly affected new shell growth.

Table 1. Effects of the test materials on shell deposition of eastern oysters exposed continuously for 96 hours in flowing, natural seawater. Shell deposition is the mean value ofshell measurements of ten oysters; standard deviations of the means are in parentheses. Test concentrationswere reported as nominal, based on additions of whole material.

Nominal concentration (µg/L)	Shell deposition (mm)	Percentage change^a
Seawater control	2.55 (0.46)	+ 28^b
Solvent control	2.00 (0.70)	---
1	2.11 (0.47)	+ 6
2	2.14 (0.84)	+ 7
4	1.11 (0.46)	- 44^b
8	0.76 (0.42)	- 62^b
16	0.47 (0.42)	- 76^b
32	0.13 (0.30)	- 94^b

a %= (Shell deposition of solvent control oysters - shell deposition of exposed oysters)/Shell deposition of solvent control oysters X 100

b Increase or decrease in new shell growth statistically significant from solvent control shell growth (P < 0.05)

Validity criteria fulfilled:

not specified

Conclusions:

Based on the findings, the 96-h EC50 was determined to be 7.5 µg/L based on nominal concentrations.

Executive summary:

The acute toxicity of the test substance to < 2 years old Eastern oyster (Crassostrea virginica) was studied in a 96-hour flow-through test. This study was conducted without following guideline, but according to "Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians". (ASTM 1980). It was compliance with GLP criteria. The oyster was exposed to five concentrations, of the test substance 0.6, 1.6, 3.2, 7.4, 15.2 and 30.8 µg/L (measured by GC, nominal concentration was 1, 2, 4, 8, 16 and 32 µg/l respectively,). Both solvent control (acetone, max. 0.1 ml/l) and control were included in this study. Nominal concentrations of > 4.0 µg/L of the substance significantly reduced new shell growth of oysters exposed to solvent only. Reduction in shell growth among exposed oysters ranged from 44% in the 4.0 µg/L test concentration to 94% in the 32 µg/L test concentration. Increases in shell growth among exposed oysters of 6 and 7% were measured for oysters exposed to 1 and 2 µg/L, respectively.

There was a significant increase in shell growth of the seawater control oysters of 28%, as compared to the solvent control oysters. It was however not expected that there was any actual solvent effect because the two lowest test concentrations also contained the same amount of acetone and were not statistically different from the seawater control.

Based on the findings, the 96-h EC50 was determined to be 7.5 µg/L based on nominal concentrations.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: EPA guideline "Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians"

Version / remarks:

1975

GLP compliance:

not specified

Vehicle:

no

Test organisms (species):

Daphnia magna

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

The highest nominal concentration of the test substance at which there was no discernible effect on the tested animals during the 48 hours toxicity test was 6.8 µg/L. After 48 hours exposuren, at test concentrations of 0.019, 0.053, 0.15 and 0.41 mg/L, 7%, 7%, 60% and 100% of mortality were observed, respectively. Table 2 represents a summary of the average observed percentage mortality of each individual test concentration after 24 and 48 hours of exposure.

Table 2. Concentrations tested and corresponding observed percentage mortalities for the water flea (Daphnia magna) exposed to the test substance.

Nominal concentration (µg/L)	Percentage mortality
	24 hour				48 hour
	A	B	C	mean	A	B	C	mean
Control (positive)	0	0	0	0	0	0	0	0
Control (negative)	0	0	0	0	0	0	0	0
6.8	0	0	0	0	0	0	0	0
19	0	0	20	7	0	0	20	7
53	0	0	0	0	0	0	20	7
150	0	20	0	7	60	60	60	60
410	100	100	100	100	100	100	100	100

Validity criteria fulfilled:

not specified

Conclusions:

Based on the findings, the 48-hours LC50 was determined to be 0.070 mg/L. The NOEC was determined to be 0.0068 mg/L.

Executive summary:

A 48-h acute static toxicity study of the test substance was performed on water flea (Daphnia magna). The study was conducted in accordance with EPA Guideline “Method for acute toxicity tests with fish, macroinvertebrates, and amphibians” (EPA, 1975), but it was not specified in compliance with GLP criteria. The daphnia were exposed to nominal concentration of 0.0068, 0.019, 0.053, 0.15 and 0.41 mg/L of the test substance (the actual concentrations were not measured). A negative control, consisting of the same dilution water and conditions, but with no test compound or acetone, was established. A positive control, consisting of the same dilution water, conditions, and containing the greatest amount of acetone present in any test vessel was also established. All test vessels were maintained at 22 ± 1°C and test solutions were not aerated during the test. Five daphnids were randomly assigned to each test vessel within 30 minutes after the compound was added for a total of 15 daphnids per concentration. During the 48 hours test, the pH and dissolved oxygen ranged from 7.2 to 7.4 and 7.9 mg/L (90% of saturation) to 8.8 mg/L (100% of saturation), respectively. The highest nominal concentration of the test substance at which there was no discernible effect on the tested animals during the 48 hours toxicity test was 0.0068 mg/L. After 48 hours exposure, at test concentrations of 0.019, 0.053, 0.15 and 0.41 mg/L, 7%, 7%, 60% and 100% of mortality were observed, respectively.

Based on the findings, the LC50 (48 h) of the test substance was determined to be 0.07 mg/L. The NOEC was determined to be 0.0068 mg/L.

Description of key information

Freshwater, 48-h LC50 = 70 µg/L, Daphnia magna, mortality, EPA guideline "Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians", LeBlanc 1977

Marine water, 96-h EC50 = 7.5 µg/L, Crassostrea virginica, growth, no guideline followed, Shults 1983

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

LC50

Effect concentration:

0.07 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

EC50

Effect concentration:

0.007 mg/L

Additional information

Freshwater

A 48-h acute static toxicity study of the test substance was performed on water flea (Daphnia magna). The study was conducted in accordance with EPA Guideline “Method for acute toxicity tests with fish, macroinvertebrates, and amphibians” (EPA, 1975), but it was not specified in compliance with GLP criteria. The daphnia were exposed to nominal concentration of 0.0068, 0.019, 0.053, 0.15 and 0.41 mg/L of the test substance (the actual concentrations were not measured). A negative control, consisting of the same dilution water and conditions, but with no test compound or acetone, was established. A positive control, consisting of the same dilution water, conditions, and containing the greatest amount of acetone present in any test vessel was also established. All test vessels were maintained at 22 ± 1°C and test solutions were not aerated during the test. Five daphnids were randomly assigned to each test vessel within 30 minutes after the compound was added for a total of 15 daphnids per concentration. During the 48 hours test, the pH and dissolved oxygen ranged from 7.2 to 7.4 and 7.9 mg/L (90% of saturation) to 8.8 mg/L (100% of saturation), respectively.

The highest nominal concentration of the test substance at which there was no discernible effect on the tested animals during the 48 hours toxicity test was 0.0068 mg/L. After 48 hours exposure, at test concentrations of 0.019, 0.053, 0.15 and 0.41 mg/L, 7%, 7%, 60% and 100% of mortality were observed, respectively.

Based on the findings, the EC50 (48 h) of the test substance was determined to be 0.07 mg/L. The NOEC was determined to be 0.0068 mg/L.

Marine water

The acute toxicity of the test substance to < 2 years old Eastern oyster (Crassostrea virginica) was studied in a 96-hour flow-through test. This study was conducted without following guideline, but according to "Standard Practice for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians". (ASTM 1980). It was compliance with GLP criteria. The oyster was exposed to five concentrations, of the test substance 0.6, 1.6, 3.2, 7.4, 15.2 and 30.8 µg/L (measured by GC, nominal concentration was 1, 2, 4, 8, 16 and 32 µg/l respectively,). Both solvent control (acetone, max. 0.1 ml/l) and control were included in this study.

Nominal concentrations of > 4.0 µg/L of the substance significantly reduced new shell growth of oysters exposed to solvent only. Reduction in shell growth among exposed oysters ranged from 44% in the 4.0 µg/L test concentration to 94% in the 32 µg/L test concentration. Increases in shell growth among exposed oysters of 6 and 7% were measured for oysters exposed to 1 and 2 µg/L, respectively.

There was a significant increase in shell growth of the seawater control oysters of 28%, as compared to the solvent control oysters. It was however not expected that there was any actual solvent effect because the two lowest test concentrations also contained the same amount of acetone and were not statistically different from the seawater control.

Based on the findings, the 96-h EC50 was determined to be 7.5 µg/L based on nominal concentrations.