o-chlorobenzenethiol

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 8th to February 10th, 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The nominal concentrations of test item used in the main experiment were: 0.013, 0.032, 0.08, 0.2 and 0.5 mg/L.
- Sampling method: Analytical measurements were performed from the control and at the applied concentration levels at the start and at the end of the experiment.
- Sample storage conditions before analysis: the samples were analysed just after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a concentration of 5.0 mg/L (nominal) was prepared with direct addition of the test item, mixed into the test medium (ISO Medium) using ultrasonic bath (approximately 3 minutes). The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the Daphnia (start of the treatments).
- Eluate: ISO medium
- Controls: ISO medium without addition of the test item. A reference control with potassium dichromate is performed at least twice a year to demonstrate satisfactory test conditions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary (definitive test).
- Age of parental stock (mean and range, SD): Less than 24 years old at the beggining of the test.
- Feeding during test: no

ACCLIMATION
- Acclimation period: None. To avoid the necessity of adaptation prior to the test, the water used in the test was similar to the culture conditions (water temperature, background colour etc.).
- Acclimation conditions (same as test or not): same as test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

248 mg/L (as CaCO3).

Test temperature:

20.3 – 20.7ºC

pH:

7.20 – 7.70

Dissolved oxygen:

8.6 – 9.2 mg/L

Nominal and measured concentrations:

- Nominal Concentrations: : 0.013, 0.032, 0.08, 0.2 and 0.5 mg/L.
- Measured geometric mean test item concentrations: 0.004; 0.012; 0.039; 0.124 and 0.318 mg/L. As the measured concentrations deviated more than 20 per cent from the nominal in most cases, biological results are based on the measured geometric mean test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Material, size, headspace, fill volume:glass, 50 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202) was
used as dilution water for both the range finding and definitive tests. The same composition of reconstitu
ted water was used for the tests and for breeding the test animals.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light/ 8 hours dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

RANGE-FINDING STUDY
Two concentration range-finding tests were conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions in both case.
- Test concentrations: 0.1, 1, 10 and 100 mg/L for the first test, and 0.005, 0.01, 0.05, 0.1 mg/L for the second test.
- Results used to determine the conditions for the definitive study: Because significant immobility was observed at the examined concentration levels during the preliminary range-finding tests and taking into account that the lowest concentration tested should preferably give no effect in the definitive test, therefore a wide concentration range and five test concentrations in a geometric series (factor 2.5) and one control were used in the main experiment under semi-static conditions. The nominal concentrations of test item used in the main experiment were: 0.013, 0.032, 0.08, 0.2 and 0.5 mg/L.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.091 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.056 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

0.124 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.039 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.124 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No abnormal behaviour or appearance of test animals was detected.
- Mortality of control: 0/20
- Other adverse effects control: none.
- Abnormal responses: no. Although 10% immobilization could be observed at the concentration of 0.012 mg/L (measured), and it was statistically significantly different from the control (even if the 24-hour data was the same and it did not show statistical significance), this difference is negligible due to that the accepted control immobilization is 10%.
Taking into account the consideration above and that no effect was observed at the test level above this concentration, the NOEC was determined as 0.039 mg/L (measured) and the LOEC could be determined as the concentration level of 0.124 mg/L (measured).
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: As the measured concentrations deviated more than 20 per cent from the nominal in most cases, biological results are based on the measured geometric mean test item concentrations. The lowest value was out of the analitically measurable range, but since the NOEC was at 0.039 mg/L, any uncertainty about the actual concentration at lower concentrations has no influence on the study interpretation.
- Effect concentrations exceeding solubility of substance in test medium: no.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- The date of the last study (Study Code: 16/281-023DA) with reference item Potassium dichromate (batch no.: A0345704) is: 14 - 15 September 2016.
- EC50 (24h): 0.93 mg/L (95 % confidence limits: 0.86 – 0.99 mg/L).

Reported statistics and error estimates:

The 24 and 48 hours EC50 values of the test item (95 % confidence limits could not be determined) were calculated using Probit analysis by SPSS PC+ software. The 48 hours EC100 values of the test item were determined directly from the raw data. For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

Table 3. Calculation of exposure concentrations.

Nominal concentration (mg/L)	Measured concentrations (mg/L) 1st renewal period		Measured concentrations (mg/L) 2nd renewal period		Geometric mean	Extrapolated value
	start	end	start	end	mg/L
Control	n.d.	n.d.	n.d.	n.d.	-	-
0.013^#	-	-	-	-	-	0.004
0.032	0.0344	0.004**	0.0382	0.004**	0.012	-
0.08	0.079	0.043	0.068	0.010*	0.039	-
0.2	0.171	0.089	0.161	0.097	0.124	-
0.5	0.406	0.252	0.400	0.251	0.318	-

n.d. – not detected

#   out of the analytically measurable range, therefore it was not measured during the whole experiment

*   concentration was below the quantification limit. In order to calculate mean value, concentration was taken as the half of the Limit of Quantification (LOQ = 0.02 mg/L) according to OECD 23; paragraph 3.3

** concentration was below the Limit of Detection. In order to calculate mean value, concentration was taken as the Limit of Detection (LOD = 0.004 mg/L) according to OECD 23; paragraph 3.3.

Since the NOEC was at 0.039 mg/L, any uncertainty about the actual concentration at lower concentrations has no influence on the study interpretation.

Table 4. Number and percentage of immobilised animals.

Concentration		Number of treated animals	Immobilised animals
Nominal	Measured		24 hours		48 hours
(mg/L)			number	percent	number	percent
Control	0.0	20	0	0	0	0
0.013	0.004	20	0	0	0	0
0.032	0.012	20	2	10	2	10*
0.08	0.039	20	0	0	0	0
0.2	0.124	20	13	65*	20	100*
0.5	0.318	20	19	95*	20	100*

*: statistically significantly different compared to the control values (Dunnett’s Test; a= 0.05)

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met. See 'overall remarks'.

Conclusions:

The 48h EC50 value of the test item on Daphnia magna is 0.091 mg/L, and the 48h NOEC is 0.039 mg/L.

Executive summary:

A short-term toxicity test on aquatic invertebrates was performed on the test item, according to OECD TG 202 / EU Method C.2, under GLP conditions. In accordance to range-finding preliminary studies, a semi-static study was performed (renewal period 24h); the nominal concentrations of test item used in the main experiment were: 0.013, 0.032, 0.08, 0.2 and 0.5 mg/L. Twenty Daphnia magna (<24 h old, 5 per replicate) were exposed to either a control medium or the test item concentrations specified for 48 hours under semi-static conditions. The test concentrations were analytically determined at the start and at the end of the renewal periods. All validity criteria were fulfilled and the analytical monitoring was performed with a validated method. As the measured concentrations deviated more than 20 per cent from the nominal in most cases, biological results are based on the measured geometric mean test item concentrations: 0.004; 0.012; 0.039; 0.124 and 0.318 mg/L.

At concentrations of 0.124 mg/L test item or higher, 100% of the daphnia were immobilised after 48h, whereas none were immobilised at 0.039 mg/L test item. Based on the results obtained, the 48h EC50 value of the test item on Daphnia magna is 0.091 mg/L, and the 48h NOEC is 0.039 mg/L.

Description of key information

Key study. Method according to OECD 202, GLP study. The 48h EC50 value of the test item on Daphnia magna is 0.091 mg/L, and the 48h NOEC is 0.039 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.091 mg/L

Additional information

Key study. Method according to OECD 202, GLP study.In accordance to range-finding preliminary studies, a semi-static study was performed (renewal period 24h); at nominal concentrations of0.013, 0.032, 0.08, 0.2 and 0.5 mg/L test item.Twenty Daphnia magna (<24 h old, 5 per replicate) were exposed to either a control medium or the test item concentrations specified for 48 hours. The test concentrations were analytically determined at the start and at the end of the renewal periods by means of a validated method. All validity criteria were fulfilled.As the measured concentrations deviated more than 20 per cent from the nominal in most cases, biological results are based on the measured geometric mean test item concentrations: 0.004; 0.012; 0.039; 0.124 and 0.318 mg/L.

At concentrations of 0.124 mg/L test item or higher, 100% of the daphnia were immobilised after 48h, whereas none were immobilised at 0.039 mg/L test item. Based on the results obtained, the 48h EC50 value of the test item on Daphnia magna is 0.091 mg/L, and the 48h NOEC is 0.039 mg/L.