3-chloro-p-toluidine

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:

30. Nov 2021 to 22. Feb 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

440/20087EEC

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC-UV

Details on sampling:

Due to the complexity of the test sample, no reference standards for chemical analysis were available. Therefore, the HPLC-UV analytical method was validated for the determination of 2-chloro-4-toluidine in test medium from daphnia tests. The parameters of linearity, specificity, accuracy, precision and limit of quantification were determined and successfully validated.
Aliquots of the samples from the biological test were directly analysed by HPLC-UV detection (range of the injection volume: 1 µL & 10 μL, depending on the expected concentration).

Vehicle:

no

Details on test solutions:

- Stock solution: 100.6 mg test item were added 1 L dilution water (stirred 2 h on a magnetic stirrer)
test item
- Test concentration: To produce the different test item concentrations ( 0.10, 0.22, 0.48, 1.1, 2.3, 5.2 und 11 mg/L (nominal)) appropriate amounts of the stock solution were diluted with dilution water to a volume of 500 mL. 50 mL of the prepared solutions were taken per replicate and 0.5 mL of dilution water containing 5 Daphnia was given to all replicates resulting in the final nominal concentrations.
- M4 medium: composition and preparation according to Elendt

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Strain of Bundesgesundheitsamt Berlin
- Age of parental stock: 20 years

ACCLIMATION
A population of parthenogenetic females of synchronized age structure has been maintained for more than 20 years in the test facility under following conditions:
- constant temperature (20 +/- 2 °C)
- 16 : 8 hour light : dark photoperiod
- light intensity: < 20 μE x m-2 x s-1
- culture water 'M4 medium') was partly renewed once a week.
- feeding: exclusively fed unicellular green algae (Desmodesmus subspicatus) 'ad libitum'.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

14.4 °dH (= 257 mg/L CaCO3), determined under non-GLP conditions

Test temperature:

18 °C to 22°C +/- 1 °C

pH:

8 +/- 1.5

Dissolved oxygen:

> 3 mg/L

Nominal and measured concentrations:

Stock solution: 100 mg/L
0.10, 0.22, 0.48, 1.1, 2.3, 5.2 and 11 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers covered with watch glasses holding 5 neonates in 50 mL test medium
- Volume of stock solution: 1L
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (so-called 'M4 medium' according to OECD 202 and EC Method C.2, annex 1, a) was used for the maintenance of the test animals and for the preparation of stock and test solutions of the test item.
- Particulate matter: no (no Tyndall effect was observed)

OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h dark

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

>= 0.22 - < 0.48 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Measured test item exposure concentrations ranged from 90.6 % to 95.0 % of their nominal target values at 0 hours and from 86.3 % to 92.2 % of their nominal target values at 48 hours.

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Measured test item exposure concentrations ranged from 90.6 % to 95.0 % of their nominal target values at 0 hours and from 86.3 % to 92.2 % of their nominal target values at 48 hours.

Details on results:

Measured test item exposure concentrations ranged from 90.6 % to 95.0 % of their nominal target values at 0 hours and from 86.3 % to 92.2 % of their nominal target values at 48 hours. The results are expressed in terms of nominal concentrations.

- Mortality of control: no mortality
- Immobilization of control: No immobilization

Reported statistics and error estimates:

The slope function is derived from the slope, b, of the linearized probit function and computes as S = 10^(1/b).

continued: Immobilisation of daphnids during exposure period

Nominal test concentration [mg/L]	Immobile daphnia
Replicate	24 h	Sum [%]	48 h	Sum [%]
Control   1	0	0	0	0
2	0		0
3	0		0
4	0		0
0.11   1	0	0	0	0
2	0		0
3	0		0
4	0		0
0.22   1	0	0	0	0
2	0		0
3	0		0
4	0		0
0.48   1	1	20	4	80
2	0		4
3	2		4
4	1		4
1.1   1	2	75	+	95
2	+		+
3	+		+
4	3		4
2.3   1	3	60	4	95
2	3		+
3	2		+
4	4		+
5.2   1	2	70	4	95
2	+		+
3	3		+
4	4		+
11   1	4	90	+	100
2	+		+
3	4		+
4	+		+

Comments: + = all Daphnia immobile

Validity criteria fulfilled:

yes

Remarks:

The immobilisation and other abnormalities in the controls did not exceed 10 % by the end of the test. The dissolved oxygen concentration remained above 3 mg/L throughout the exposure period and the pH of exposure solutions did not change by more than ± 1

Conclusions:

The acute toxicity of "3-chloro-4-methylaniline" to Daphnia magna was evaluated in a static test according to Council Regulation (EC) No. 440/2008, Method C.2 "Acute toxicity to Daphnia", which is in accordance with OECD Guideline for the Testing of Chemicals No. 202 "Daphnia sp., Acute Immobilization Test".
Swimming ability and percentage of immobilization were observed. After 48 hours of exposure, values between ≥ 0.22 < 0.48 were determined. Measured exposure concentrations of test items ranged from 90.6% to 95.0% of their nominal target values at 0 hours and from 86.3% to 92.2% of their nominal target values at 48 hours.

Executive summary:

A study was performed to assess the acute toxicity of 2-Chlor-4-toluidin to Daphnia magna STRAUS under static conditions. The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.2 ‘Acute toxicity for Daphnia’ (2008) which is equivalent to OECD Guideline for Testing of Chemicals No. 202 'Daphnia sp., Acute Immobilisation Test' (adopted April 13, 2004). The Daphnia were exposed to a range of concentrations of nominally 0.10, 0.22, 0.48, 1.1, 2.3, 5.2 and 11 mg/L of 2-Chlor-4-toluidin dissolved in dilution water. Auxiliary used to prepare the test media was a magnetic stirrer. Observations were made on the swimming ability and the percentage immobilised, respectively, after 24 and 48 hours of exposure. The following values were determined:

EC₅₀ (24 h) = 1.5 mg/L

EC₅₀ (48 h) ≥ 0.22 < 0.48

The results are expressed in terms of nominal concentrations. Measured test item exposure concentrations ranged from 90.6 % to 95.0 % of their nominal target values at 0 hours and from 86.3 % to 92.2 % of their nominal target values at 48 hours.

Description of key information

A study was performed to assess the acute toxicity of 2-Chlor-4-toluidin to Daphnia magna STRAUS under static conditions. The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.2 ‘Acute toxicity for Daphnia’ (2008) which is equivalent to OECD Guideline for Testing of Chemicals No. 202 'Daphnia sp., Acute Immobilisation Test' (adopted April 13, 2004). The Daphnia were exposed to a range of concentrations of nominally 0.10, 0.22, 0.48, 1.1, 2.3, 5.2 and 11 mg/L of 2-Chlor-4-toluidin dissolved in dilution water. Auxiliary used to prepare the test media was a magnetic stirrer. Observations were made on the swimming ability and the percentage immobilised, respectively, after 24 and 48 hours of exposure. The following values were determined:

EC50 (24 h) = 1.5 mg/L

EC50 (48 h) ≥ 0.22 < 0.48

The results are expressed in terms of nominal concentrations. Measured test item exposure concentrations ranged from 90.6 % to 95.0 % of their nominal target values at 0 hours and from 86.3 % to 92.2 % of their nominal target values at 48 hours.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Remarks:

48 h

Effect concentration:

>= 0.22 - < 0.48 mg/L