Chlorothalonil

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Nov 2003 to 5 Dec 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the test media of all test concentrations with surviving fish and from the solvent control were taken immediately before the start of the test (0 hours), after 48 hours and at the end of the test (after 96 hours). However, from the highest nominal concentration of 100 µg/L samples were taken after 24 hours, since all fish were dead at that date.
All test medium samples were taken from the approximate center of the aquaria without mixing of the test media and were deep frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (without GLP) the test item is sufficiently stable in the test water under the storage conditions.

Vehicle:

yes

Remarks:

N,N-Dimethylformamide

Details on test solutions:

The solvent N,N-Dimethylformamide (= DMF) was used as vehicle for the dosage of the test item with limited water solubility. This solvent was chosen for its solubility properties and its relative non toxicity to fish. The DMF concentration in the test media was 100 µL/L. According to a pre-experiment (without GLP), the test item was stable in N,N-Dimethylformamide over the application solution renewal period of 4 days.
The application solutions used for the dosage of the different test media during the four day test duration were freshly prepared on Day 0 (start of the test). The application solution used for the dosage of the highest test concentration of nominal 100 µg/L was prepared by dissolving 400 mg of the test item in 400 mL DMF by means of stirring for 15 minutes at room temperature. Adequate volumes of this application solution were diluted with DMF to prepare the application solutions used for the dosage of the lower test concentrations.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Length at study initiation: 5.6 ± 0.23 cm
- Weight at study initiation: 1.7 ± 0.16 g

ACCLIMATION
- Acclimation period anc condition: Held in the laboratories of the test facility for more than two weeks without any medication. Prior to test start, they were acclimated for one week to the test water and temperature.
- Feeding: During holding and acclimation, until one day before the start of the test, the fish were fed with a commercial fish diet. No feeding during the test period.
- Health during acclimation (any mortality observed): During holding and acclimatization no fish died in the test fish batch and all fish were healthy.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

13.0 - 13.8 °C

pH:

7.9 - 8.0

Dissolved oxygen:

9.3 - 9.9 mg O2/L

Nominal and measured concentrations:

- Nominal concentration: 4.6, 10, 22, 46, and 100 µg/L
- Measured concentration: 4.99, 11.1, 23.1, 45.9 and 89.3 µg/L, respectively. See Table 1 in "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 L flow-through aquarium
- Material, size, headspace, fill volume: Length: 54 cm; wide: 29 cm, height: 40 cm; height of water level: 31 cm; with 48 liter water volume
- Aeration: Yes
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- No. of vessels per vehicle control: 1
- Biomass loading rate: 0.25 g fish wet weight per liter test medium (based on the volume of the aquarium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Local tap water (non chlorinated well water of drinking water quality), reduced for total hardness by ion exchange.
- Total hardness of test water: 216 mg/L as CaCO3
- The test water was aerated prior to the preparation of the test media until oxygen saturation was reached.
- Intervals of water quality measurement: The pH-value, oxygen concentration and water temperature were measured in each test medium, the solvent control and the control prior to the introduction of the fish and thereafter once every day during the test. The total hardness of the water used in the test was measured at the start of the test in the control. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity: Approximately 230-430 Lux (with a 30-minute transition period)

EFFECT PARAMETERS MEASURED:
-The behaviour and survival of the fish were assessed at 3, 24, 48, 72 and 96 hours after initiation of the test.

TEST CONCENTRATIONS
- The test concentrations were based on the results of a range-finding test and the results of a pre-experiment to determine the dosage of the test item in the test medium.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

39 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

22 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

An overview of the results is provided in Table 2 in "Any other information on results incl. tables".

In the control, the solvent control and at the test concentrations up to and including 22 µg/L all fish survived until the end of the test and no visible abnormalities were observed at the test fish. At the next higher test concentration of 46 µg/L all test fish showed strong visible abnormalities one day after test start, and at the end of the test six of the seven test fish had died at this test concentration. At the highest concentration of 100 µg/L all fish were dead already after one day test duration.

Sublethal observations / clinical signs:

Table 2. Results (7 fish tested at each treatment )

Nominal concentration (µg/L)	Number of affected fish* / number of dead fish visible abnormalities
	3 h	24 h	48 h	72 h	96 h
Control	0/0	0/0	0/0	0/0	0/0
Solvent control	0/0	0/0	0/0	0/0	0/0
4.6	0/0	0/0	0/0	0/0	0/0
10	0/0	0/0	0/0	0/0	0/0
22	0/0	0/0	0/0	0/0	0/0
46	0/0	7/0	7/6	7/6	7/6
100	0/0	7/7	-/-	-/-	-/-
LC50values (µg/L)	>100	68	39	39	39
95% confidence limits	-	-	20 - 75	20 - 75	20 - 75

* : Number of dead fish plus number of fish with intoxication symptoms

-/- : all fish dead

Validity criteria fulfilled:

yes

Remarks:

See validation criteria in Ány other information on materials and methods'

Conclusions:

Based on the findings, the 96-hour NOEC of the test substance to rainbow trout was determinded to be 22 µg/L and the LC50 was calculated to be 39 µg/L.

Executive summary:

The acute toxicity of the test substance to rainbow trout (Oncorhynchus mykiss) was studied in a 96-hour flow-through test. The test was conducted according to OECD TG 203 and EU Method C1. It was also compliance with GLP criteria. The fish were exposed to theN,N Dimethylformamide dissolved compound at the mean measured concentrations of 4.99, 11.1, 23.1, 45.9 and 89.3 µg/L (measured by HPLC; the nominal concentrations were4.6, 10, 22, 46 and 100 µg/L, respectively). But the nominal concentration was used to report the biological effects. There were negative control and carrier control in the test as well. The test was carried out under the following conditions: pH 7.9 – 8.1 and dissolved oxygen concentration 9.3 - 9.9 mg O2/L (> 60% of saturation). The loading rate was 0.25 g fish per L test medium.

In the control, the solvent control and at the test concentrations up to and including 22 µg/L all fish survived until the end of the test and no visible abnormalities were observed at the test fish. At the next higher test concentration of 46 µg/L all test fish showed strong visible abnormalities one day after test start, and at the end of the test six of the seven test fish had died at this test concentration. At the highest concentration of 100 µg/L all fish were dead already after one day test duration.

Based on the findings, the 96-hour NOEC of the test substance to rainbow trout amounted to be 22 µg/L. The 96-hour LC50 was calculated to be 39 µg/L.

 

 

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

Draft

GLP compliance:

yes

Test organisms (species):

Cyprinodon variegatus

Test type:

semi-static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

28 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

20 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC10

Effect conc.:

21 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

- The measured concentrations are shown in Table 1. The limit of quantification in this study was 0.0268 μg/L. Geometric mean measured concentrations were used for the calculation and reporting of results.

- The LC10, LC20 and LC50 values are defined as the concentration of the test substance in dilution water which cause mortality of 10%, 20% and 50%, respectively, of the test organism population at the stated time interval. CETISTM Version 1.8 was used to perform the statistical computations. If no concentration tested caused a reduction of 50% survival, the LC50 value was empirically estimated to be greater than the highest geometric mean measured concentration tested and no statistical analyses were performed.

- The NOEC was also determined. The mortality data and estimated LC50 values are shown in the Table 2.

Table 1. Analytical results

Nominal concentration (µg/L)	Measured Concentration (μg/L)				Geometric mean measured concentration (µg ai/L)
	0 hours	48 hours (aged)	48 hours (new)	96 hours
6.3	5.4	3.4	6.1	3.5	4.5
13	12	6.9	11	6.0	8.6
25	23	17	24	16	20
50	45	27	48	37	38
100	110	120	nm	nm	110

NM= Not Measured. Test solution was not sampled due to 100% mortality in the treatment level.

Table 2. Effects of the test substance on the survival of sheepshead minnow (Cyprinodon variegatus)

Geometric mean measured concentration (µg/L)	Mortality observed (Cumulative number of dead fish) (n = 7)
	24 hours	48 hours	72 hours	96 hours
Dilution water control	0	0	0	0
Solvent control	0	0	0	0
4.5	0	0	0	0
8.6	0	0	0	0
20	0	0	0	0
38	0#	7	7	7
110	7	7	7	7
LC50 µg/L	65	28
LC20 µg/L	47	23
LC10 µg/L	42	21
NOEC µg/L	38	20

# Fish were observed to be on the bottom of the test vessel, were lethargic and had loss of equilibrium

Validity criteria fulfilled:

yes

Remarks:

See validity criteria in 'Any other information on materials and methods incl. tables'

Conclusions:

Based on geometric mean measured concentrations, the 96-hour LC50 for the test substance to sheepshead minnow (Cyprinodon variegatus) was 28 µg/L and the 96-hour NOEC was 20 µg/L. The LC10 and LC20 were 21 and 23 µg/L, respectively.

Executive summary:

This study was conducted to determine the acute effects of the test substance on the sheepshead minnow, Cyprinodon variegatus, under static-renewal conditions following OCSPP Draft Guideline 850.1075 and OECD TG 203. It was compliance with GLP criteria. One aquarium, containing seven fish each, was established for the controls and the 6.3, 13, 25, 50 and 100 µg/L treatment levels (4.5, 8.6, 20, 38 and 110 µg/L as geometric mean measured concentrations) and was maintained in a temperature-controlled water bath at 21 to 23 ºC with a 16 hour light: 8 hour dark photoperiod at a light intensity of 74 to 81 footcandles (800 to 870 lux). Test solutions were renewed at the 48-hour interval. Aquaria were examined at 0, 6, 24, 48, 72 and 96 hours of exposure as follows: dead fish were recorded and removed, biological observations, including adverse effects (e.g., loss of equilibrium) of the exposed sheepshead minnow and observations of the physical characteristics of the test solutions (e.g., presence of precipitate, film on the solutions’ surface) were made and recorded. Effects for this study were based on death, defined as the lack of movement by the exposed organisms (i.e., absence of gill movement and reaction to gentle prodding).

At the 6-hour observation interval, no mortalities were recorded, however, fish were observed to be lethargic in the 110 µg/L treatment level. After 24-hours of exposure, 100% mortality was observed in the 110 µg/L treatment level and sub-lethal effects were observed in the 38 µg/L treatment level. At the 48-hour interval, 100% mortality was observed among fish exposed to the 38 µg/L treatment level. After 96−hours of exposure, no mortality or sublethal effects were observed among fish exposed to any of the remaining treatment levels tested (4.5, 8.6 and 20 µg/L) or the controls. Based on geometric mean measured concentrations, the 96-hour LC50 value was determined using linear interpolation to be 28 µg/L and no confidence intervals could be calculated.The NOEC for this study was determined to be 20 µg/L.

Description of key information

Freshwater, 96-h LC50 = 39 µg/L, Oncorhynchus mykiss, OECD TG 203, Peither 2003

Marine water, 96-h LC50 = 28 µg/L, Cyprinodon variegatus, mortality, OECD TG 203, Fournier 2013

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

39 µg/L

Marine water fish

Marine water fish

Dose descriptor:

LC50

Effect concentration:

28 µg/L

Additional information

Freshwater

The acute toxicity of the test substance to rainbow trout (Oncorhynchus mykiss) was studied in a 96-hour flow-through test. The test was conducted according to OECD TG 203 and EU Method C1. It was also compliance with GLP criteria. The fish were exposed to the N,N Dimethylformamide dissolved compound at the mean measured concentrations of 4.99, 11.1, 23.1, 45.9 and 89.3 µg/L (measured by HPLC; the nominal concentrations were4.6, 10, 22, 46 and 100 µg/L, respectively). But the nominal concentration was used to report the biological effects. There were negative control and carrier control in the test as well. The test was carried out under the following conditions: pH 7.9 – 8.1 and dissolved oxygen concentration 9.3 - 9.9 mg O2/L (> 60% of saturation). The loading rate was 0.25 g fish per L test medium.

In the control, the solvent control and at the test concentrations up to and including 22 µg/L all fish survived until the end of the test and no visible abnormalities were observed at the test fish. At the next higher test concentration of 46 µg/L all test fish showed strong visible abnormalities one day after test start, and at the end of the test six of the seven test fish had died at this test concentration. At the highest concentration of 100 µg/L all fish were dead already after one day test duration.

Based on the findings, the 96-hour NOEC of the test substance to rainbow trout amounted to be 22 µg/L. The 96-hour LC50 was calculated to be 39 µg/L.

 

Marine water

This study was conducted to determine the acute effects of the test substance on the sheepshead minnow, Cyprinodon variegatus, under static-renewal conditions following OCSPP Draft Guideline 850.1075 and OECD TG 203. It was compliance with GLP criteria. One aquarium, containing seven fish each, was established for the controls and the 6.3, 13, 25, 50 and 100 µg/L treatment levels (4.5, 8.6, 20, 38 and 110 µg/L as geometric mean measured concentrations) and was maintained in a temperature-controlled water bath at 21 to 23ºC with a 16 hour light: 8 hour dark photoperiod at a light intensity of 74 to 81 footcandles (800 to 870 lux). Test solutions were renewed at the 48-hour interval. Aquaria were examined at 0, 6, 24, 48, 72 and 96 hours of exposure as follows: dead fish were recorded and removed, biological observations, including adverse effects (e.g., loss of equilibrium) of the exposed sheepshead minnow and observations of the physical characteristics of the test solutions (e.g., presence of precipitate, film on the solutions’ surface) were made and recorded. Effects for this study were based on death, defined as the lack of movement by the exposed organisms (i.e., absence of gill movement and reaction to gentle prodding).

At the 6-hour observation interval, no mortalities were recorded, however, fish were observed to be lethargic in the 110 µg/L treatment level. After 24-hours of exposure, 100% mortality was observed in the 110 µg/L treatment level and sub-lethal effects were observed in the 38 µg/L treatment level. At the 48-hour interval, 100% mortality was observed among fish exposed to the 38 µg/L treatment level. After 96−hours of exposure, no mortality or sublethal effects were observed among fish exposed to any of the remaining treatment levels tested (4.5, 8.6 and 20 µg/L) or the controls.

Based on geometric mean measured concentrations, the 96-hour LC50 value was determined using linear interpolation to be 28 µg/L and no confidence intervals could be calculated. The NOEC for this study was determined to be 20 µg/L.