2-Amino-5-Chloro-N,3-Dimethylbenzamide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 2020 to 6 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: 2-amino-5-chloro-N,3-dimethylbenzamide
Batch/Lot Number: 628-042-00
Test item content: 983.7 ± 2.8 g/kg
Physical State: Beige Solid
Date of certificate of analysis: 09th March 2020
Date of Expiry: 17 October 2021
Storage Conditions: Dark, room temperature (15-25°C), < 70 % relative humidity

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal 0.123, 0.370, 1.110, 3.333 and 10.00 mg test item/L in EPA’s AAP growth medium containing 0.01 % DMSO as solvent.
- Sampling method: five treated samples, two untreated samples. Three replicates were prepared for the treated groups and six replicates for the control groups, each replicate containing 100 mL of solution.
- The concentration of the test item was determined by UPLC-MS/MS analyses, in samples of the test solutions at test start, after 24h, 48h and at test end after 72 h.
- Sample storage conditions before analysis: The samples were stored deep-frozen at ≤ -18°C prior to analysis. Analysis was performed within one week after termination of the biological part of the experiment.
- The samples to be analysed were collected as follows:
- 7 samples at 0 hours, from the fresh solutions: 2 untreated controls, 5 concentrations of test item;
- 7 samples at 24 hours, from the exposed solutions: 2 untreated controls, 5 concentrations of test item;
- 7 samples at 48 hours, from the exposed solutions: 2 untreated controls, 5 concentrations of test item;
- 7 samples at 72 hours, from the aged solutions: 2 untreated controls, 5 concentrations of test item.
The samples were diluted with Diluent before the analysis. Each sample was measured by double injection and the content of test item in the test samples was calculated on the basis of the calibration curve equation obtained in the same chromatographic session.

Vehicle:

no

Details on test solutions:

- 11.1 mg of analytical standard (= test item) were weighed in a 10 mL graduated flask and made up to volume with DMSO, to obtain the SS1 solution at 1091.9070 mg a.i./L;
- 1.00 mL of SS1 solution was diluted with DMSO, in a 10 mL graduated flask, to obtain the SS2 solution at 109.1907 mg a.i./L;
- 1.00 mL of SS2 solution was diluted with EPA medium, in a 10 mL graduated flask, to obtain the SS3 solution at 10.9191 mg a.i./L;
- 0.10 mL of SS3 solution were diluted with EPA medium, in a 10 mL graduated flask, to obtain the SS4 solution at 109.1907 μg a.i./L.
The solutions for the linearity control were prepared in graduated flasks and made up to volume with EPA medium and appropriate aliquots of solvent, to have a final concentration of 0.01% of DMSO. Each solution was analysed with two injections.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (new name: Raphidocelis subcapitata)
- Strain: SAG 61.81
- Source (laboratory, culture collection): In-house culturing at the Test Facility, originating from SAG Culture collection of algae (Germany).
Pseudokirchneriella subcapitata is maintained in an incubator, at a temperature in the range of 23±2°C and with continuous light (intensity in the range of 4440-8880 Lux), in AAP medium (US EPA, OECD 201).
The algae cultures are periodically prepared by inoculating the fresh growth medium with algal cells (104 cells/mL) from older cultures.
The culture utilized for the inoculums of this test was prepared 4 days before the test beginning (code of the culture: Ps 12/03/20).

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21.4 - 23.9 °C

pH:

7.40 - 7.55 in the untreated control groups and 7.04 - 7.38 in the treatment groups.

Nominal and measured concentrations:

Nominal concentrations: 0.123, 0.370, 1.110, 3.333 and 10.00 mg test item/L

Details on test conditions:

TEST SYSTEM
- thermostatic chamber
- Initial cells density: 104 cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): three replicates for each test concentration
- No. of vessels per control (replicates): six replicates for the untreated control groups

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: measured at the beginning and at the end of the study
- Light intensity and quality: continuous and in the range of 5961 - 6796 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The biomass in each flask and the appearance of the cells were assessed daily during the test period, by electronic particle counter (each sample was evaluated with three counts and the cell density was expressed as mean of the three obtained values).
Microscopic observations were performed in order to assess any abnormal appearance of the algal cells that may have been caused by exposure to the test item.

TEST CONCENTRATIONS
- Test concentrations: 0.123, 0.370, 1.110, 3.333 and 10.00 mg test item/L

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Growth inhibition based on yield: EyC50

Effect conc.:

0.276 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

Corresponding LOEyC

Effect conc.:

0.331 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Corresponding NOEyC

Effect conc.:

0.065 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

ErC50

Effect conc.:

1.291 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

Corresponding LOErC

Effect conc.:

0.065 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Corresponding NOErC

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no

Reported statistics and error estimates:

The determination of the ECx values was performed by Probit analysis using linear maximum likelihood regression.
Williams multiple sequential t-test procedure and Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm were used to estimate the LOEC/NOEC values at 72 hours (for growth rate and yield respectively).
There were no significant statistical differences between control and solvent control, therefore the treated groups were compared with the solvent control.
The assessment of the effects was based on the geometric mean measured concentrations of M198. The ECx endpoints, with 95% confidence limits, the lowest observed effect concentrations (LOEC) and the no observed effect concentrations (NOEC), are reported.
For yield, data was normally distributed (Shapiro-Wilk’s Test) but variance homogeneity was not given (Levene’s Test), thus a Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm (α = 0.05, one-sided smaller) was applied.
For growth rate, data was normally distributed (Shapiro-Wilk’s Test) and variance homogeneity given (Levene’s Test) a Williams Multiple Sequential t-test was applied (α = 0.05, one-sided smaller).

Summary of the analytical results for fresh and aged media samples from the biological part of the study withPseudokirchneriella subcapitata:

Code	Nominal test item conc. [mg/L]	Nominal conc. [mg a.i./L]	Geometric mean of measured conc. [mg a.i./L]
CTRL	0.00	0.00	< LOD *
CT SOLV	0.00	0.00	< LOD *
C1	0.123	0.121	0.017
C2	0.370	0.364	0.065
C3	1.110	1.093	0.331
C4	3.333	3.279	1.429
C5	10.00	9.837	4.726

* Parameter from the validated analytical method: LOD 0.0546 μg/L; LOQ 0.1 μg/L.

The analytical determination of the content of active ingredient in the test solutions showed recovery values not within the range 80-120% of the nominal values (with a mean recovery of 93.12% in the fresh samples and 38.47 % after 24 h, 32.12 % after 48 h and 9.47% in the 72 h aged samples, therefore the data evaluation was performed using the geometric mean measured concentrations.

 

The percentage of inhibition of growth based on growth rate (%Ir) and on yield (%Iy) for each test concentration and any significant inhibition compared to the control (after 72 hours) are reported in the Table with a summary of the analytical results for the biological samples from the current study:

 

Inhibition of growth ofPseudokirchneriella subcapitatabased on growth rate (Ir) and yield (Iy) in percent after 72 h-exposure to the Test item, in relation to the control:

Code	Nominal conc. [mg a.i./L]	Measured conc. [mg a.i./L]	%Ir^A	%Iy^B
C1	0.123	0.017	0.33	1.66
C2	0.370	0.065	3.11 +	13.21
C3	1.110	0.331	17.15^+	54.74^+
C4	3.333	1.429	52.58^+	91.76^+
C5	10.00	4.726	81.85^+	98.61^+

A⁺indicates a significant difference compared to the untreated control (Williams Multiple Sequential t-test, α = 0.05, one-sided smaller)

B⁺indicates a significant difference compared to the untreated control (Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm, α = 0.05, one-sided smaller)

The initial cell density was 10⁴cells/mL. Growth ofPseudokirchneriella subcapitatais inhibited in a concentration-dependent manner, showing an increasing inhibition with increasing test concentration when compared to the untreated control.

For the calculation of effect concentrations below, the treatments were compared with the solvent control.

 

Effect concentrations of the Test item for growth rate and yield of Pseudokirchneriella subcapitata after 72 h exposure (based on geometric mean measured M198. concentrations):

Endpoint (72 hours)	Geometric mean meas. concentration [mg a.i./L]	Confidence limits		Endpoint (72 hours)	Geometric mean meas. concentration [mg a.i./L]	Confidence limits
		Lower	Upper			Lower	Upper
EyC10	0.055	0.044	0.067	ErC10	0.202	0.184		0.220
EyC20	0.096	0.082	0.111	ErC20	0.381	0.356		0.407
EyC50	0.276	0.251	0.304	ErC50	1.291	1.240		1.343
LOEyC	0.331	-	-	LOErC	0.065	-		-
NOEyC	0.065	-		NOErC	0.017	-		-

Validity criteria results of the growth inhibition test with Pseudokirchneriella subcapitata:

VALIDITY CRITERIA	Threshold	Observed in the solvent control
Cells concentration in the control cultures within 3 days:	Increased by a factor of at least 16 (specific growth rate at least 0.92 day^-1)	Factor 92.96 (mean specific growth rate 1.510 day^-1)
Mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures:	Not exceed 35%	18.8%
Coefficient of variation of average specific growth rates during the whole test period in the replicate of the control group:	Not exceed 7%	1.5%

Validity criteria fulfilled:

yes

Conclusions:

The growth inhibition test of the test item 2-amino-5-chloro-N,3-dimethylbenzamide on green alga Pseudokirchneriella subcapitata, after 72 hours of exposure gave the following results:
- Growth inhibition based on yield: EyC50 = 0.276 mg a.i./L (95 % C.L. 0.251 - 0.304 mg a.i./L);
- Corresponding LOEyC = 0.331 mg a.i./L and NOEyC = 0.065 mg a.i./L;
- Growth inhibition based on growth rate: ErC50 = 1.291 mg a.i./L (95 % C.L. 1.240 - 1.343 mg a.i./L);
- Corresponding LOErC = 0.065 mg a.i./L and NOErC = 0.017 mg a.i./L.

Based on EU regulation 1272/2008 the results lead to classification as hazardous to aquatic environment, chronic category 2 (H411 Toxic to aquatic life with long lasting effects.)

Executive summary:

The growth inhibition test was performed in order to evaluate the effects of the test item 2-amino-5-chloro-N,3-dimethylbenzamide (M198) on green alga Pseudokirchneriella subcapitata, after 72 hours of exposure.

The cultures were exposed to nominal 0.123, 0.370, 1.110, 3.333 and 10.00 mg test item/L, plus two untreated control groups (with and without solvent).

All test item concentrations and controls were analysed at test start and after 24h, 48h and at test end after 72 h. The analytical determination of the active ingredient content showed a mean recovery of 93.12% in the fresh samples, 38.47 % after 24 h, 32.12 % after 48 h and 9.47% in the 72 h aged samples. Accordingly, the assessment of the effects was based on the geometric mean measured concentrations of the active ingredient, which were calculated to be as follows: 0.017, 0.065, 0.331, 1.429 and 4.726 mg a.i./L.

After 72 hours exposure, no cells with abnormal appearance were observed in any of the treatment groups.

The endpoints, based on the geometric mean measured concentrations of active ingredient, were estimated to be the following:

- Growth inhibition based on yield: E_yC₅₀= 0.276 mg a.i./L(95 % C.L. 0.251 - 0.304 mg a.i./L);

- CorrespondingLOE_yC = 0.331 mg a.i./L and NOE_yC = 0.065 mg a.i./L;

- Growth inhibition based on growth rate:E_rC₅₀= 1.291 mg a.i./L(95 % C.L. 1.240 - 1.343 mg a.i./L);

- CorrespondingLOE_rC = 0.065 mg a.i./L and NOE_rC = 0.017 mg a.i./L.

 

No cells with abnormal appearance were observed in any of the control or test item treatment groups.

 

The validity criteria of OECD 201 (2011) were fulfilled, thus the study is valid.

 

Description of key information

- Growth inhibition based on yield: E_yC₅₀= 0.276 mg a.i./L(95 % C.L. 0.251 - 0.304 mg a.i./L);

- Corresponding LOE_yC = 0.331 mg a.i./L and NOE_yC = 0.065 mg a.i./L;

- Growth inhibition based on growth rate:E_rC₅₀= 1.291 mg a.i./L (95 % C.L. 1.240 - 1.343 mg a.i./L);

- Corresponding LOE_rC = 0.065 mg a.i./L and NOE_rC = 0.017 mg a.i./L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.276 mg/L

EC10 or NOEC for freshwater algae:

0.017 mg/L