3-chloro-p-toluidine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Nov 2021 to 24 Mar 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

761/2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

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:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution: 99.9 mg test item were added 1 L dilution water (stirred 24 h on a magnetic stirrer). The solution was dissolved and no particles were still visible (no Tyndall effect was observed).
- Controls: yes
- Test item concentrations: 0.010, 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (nominal)
- Test concentration separation factor: 3.2
- Other relevant information: To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 500 mL. 100 mL of the prepared solutions were taken per replicate and 1.04 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: No. 86.81 SAG
- Source (laboratory, culture collection): 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).
- Method of cultivation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a particle counter, Z2, Beckmann Coulter.

ACCLIMATION
- Acclimation period: 3 days in OECD medium of OECD TG 201

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

1.3 °dH (= 22.5 mg/L CaCO3)

Test temperature:

21 °C to 24 °C +/- 2 °C
Deviations : A short-term temperature rise at the beginning of the test to 24.1 °C for approx. 2 hours of exposure had no influence on the test results. As the temperature was otherwise within the mandatory range for the entire duration of the test.

pH:

8.0 to 8.6

Nominal and measured concentrations:

Stock solution: 100 mg/L
0.010, 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with cotton stoppers
- Type (delete if not applicable): closed
- Material, fill volume: glass, 100 mL
- Initial cells density: 5000
- Control end cells density: measured in a particle counter (Z2, Beckmann Coulter) by taking small aliquots from each test flask, which were not replaced.
- No. of organisms per vessel: 5000
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity: 4000 - 8000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: particle counter (Z2, Beckmann Coulter)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

Range finding study: yes
- Test concentrations (range finding): 0.010, 0.10, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: yes

CULTURING APPARATUS
-Details on culturing apparatus used:

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Measured test item concentrations ranged from 98.2 % to 104.7 % of their nominal target values at 0 hours, and from 94.3 % to 99.8 % of nominal target values at 72 hours.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Measured test item concentrations ranged from 98.2 % to 104.7 % of their nominal target values at 0 hours, and from 94.3 % to 99.8 % of nominal target values at 72 hours.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The NOEC could not be performed due to mathematical reasons. Therefore, the values were read directly from the results.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The LOEC could not be performed due to mathematical reasons. Therefore, the values were read directly from the results.

Details on results:

Measured test item concentrations ranged from 98.2 % to 104.7 % of their nominal target values at 0 hours, and from 94.3 % to 99.8 % of nominal target values at 72 hours. The results are expressed in terms of nominal concentrations.
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no particles; no Tyndall effect was observed
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

The 72 hour EC 10 and EC 50 were calculated by probit analysis. All calculations were carried out using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).
The slope function is derived from the slope, b, of the linearized probit function and computes as S = 10^(1/b).

Effective Concentrations (ECx) for Growth Rate at 72 h of Desmodesmus subspicatus
Table below: %Inhibition caused by the test item after 72 h.

Treatm.[mg/L]	mean	std. dev.	n	% inhibition
control	1.867	0.0485	3	0.0
0.010	1.862	0.0451	3	0.2
0.032	1.835	0.0326	3	1.7
0.100	1.823	0.0137	3	2.3
0.320	1.866	0.0203	3	0.0
1.000	1.759	0.0859	3	5.8
3.200	1.712	0.0154	3	8.3
10.000	1.370	0.0370	3	26.6
32.000	0.251	0.1621	3	86.6
100.000	0.009	0.0133	3	99.5

For mathematical reasons, NOEC/LOEC determinations for growth rate could not be performed. In a regulatory context, EC 10 values are generally considered a surrogate for NOEC. In the present case, a 10% effect on growth rate is caused by a concentration of > 3.2 mg/L and < 10 mg/L, therefore NOEC and LOEC were read directly from the table above (NOEC = 3.2 mg/L and LOEC = 10 mg/L).

Validity criteria fulfilled:

yes

Remarks:

The cell density in the control cultures increased by a factor of at least 16 within 72 hours. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 7 %.

Conclusions:

The acute toxicity of "3-chloro-4-methylaniline” with Desmodesmus subspicatus was evaluated in a static test according to Council Regulation (EC) No. 761/2009, Method C.3 "Freshwater Alga and Cyanobacteria, Growth inhibition test ", which is in accordance with OECD Guideline for the Testing of Chemicals No. 201 "Alga, Growth Inhibition Test ".
Exponentially growing algal cells were determined over a 72 h period, the following values were obtained: ErC 50 (72 h) = 15 mg/L and ErC 10 (72 h) = 6.1 mg/L. Measured test item concentrations ranged from 98.2 % to 104.7 % of their nominal target values at 0 hours, and from 94.3 % to 99.8 % of nominal target values at 72 hours.

Executive summary:

A study was performed to assess the adverse effects of 2-Chlor-4-toluidin on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a ange of concentrations, nominally 0.010, 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of 2-Chlor-4-toluidin dissolved in dilution water. Auxiliary used to prepare the test media was a magnetic stirrer. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r) and yield (y), relative to control cultures grown under identical conditions. The following values were determined:

ErC 50 (0-72 h): 15
ErC 10 (0-72 h): 6.1
The results are expressed in terms of nominal concentrations. Measured test item concentrations ranged from 98.2 % to 104.7 % of their nominal target values at 0 hours, and from 94.3 % to 99.8 % of nominal target values at 72 hours.

Description of key information

A study was performed to assess the adverse effects of 2-Chlor-4-toluidin on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations. The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours to a ange of concentrations, nominally 0.010, 0.032, 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L of 2-Chlor-4-toluidin dissolved in dilution water. Auxiliary used to prepare the test media was a magnetic stirrer. The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r) and yield (y), relative to control cultures grown under identical conditions. The following values were determined:

ErC 50 (0-72 h): 15
ErC 10 (0-72 h): 6.1
NOEC [r]: 3.2
LOEC [r]: 10

The results are expressed in terms of nominal concentrations. Measured test item concentrations ranged from 98.2 % to 104.7 % of their nominal target values at 0 hours, and from 94.3 % to 99.8 % of nominal target values at 72 hours.

Key value for chemical safety assessment

EC50 for freshwater algae:

15 mg/L

EC10 or NOEC for freshwater algae:

6.1 mg/L