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

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:

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: 2-Amino-5-Chloro-N,3-Dimethylbenzamide (M198)
Batch: 628-042-00
CAS number: [890707-28-5]
Test item content: 983.7 ± 2.8 g/kg
Analysis date: 16 October 2019
Expiry date: 16 October 2021
Supplier: ADAMA Makhteshim Ltd.
Appearance: Beige powder
Packaging: White opaque plastic bag
Quantity: received 1 kg
Date of receipt: 12 November 2019
Storage: In darkness at room temperature

Analytical monitoring:

yes

Details on sampling:

Concentration/control:
A semi-static concentration-response test was conducted at 6.25 - 12.50 - 25.00 - 50.00 - 100.00 mg/L of test item in ISO medium containing 0.01 % of the solvent DMSO, plus two untreated control groups (with and without solvent).

Sampling method:
20 daphnids, divided in 4 replicates of 5 daphnids, were used for each group.
The test organisms were exposed to the test solutions for 48 hours, with renewal of the test solutions after 24 hours. The number of mobile and immobile daphnids was counted at 24 and 48 hours after the beginning of the test.

Sample analysis:
The samples to be analysed were collected as follows: at the start of the test from fresh test solutions (T0), at the renewal from the freshly prepared and the spent test solutions (T24), as well as at the end of the test from the spent test solutions (T48); the spent samples were taken after the pooling of the replicates. 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 aged solutions: 2 untreated controls, 5 concentrations of test item;
- 7 samples at 24 hours, from the fresh solutions: 2 untreated controls, 5 concentrations of test item;
- 7 samples at 48 hours, from the aged solutions: 2 untreated controls, 5 concentrations of test item.
The samples were diluted to the calibration range with Diluent before the analysis.
Each sample was measured by double injection and the content of active ingredient 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:

All stock and test solutions were prepared in ISO medium containing 100 μL/L solvent (DMSO, 0.01%).
At the start of the test, a stock solution of 100 mg test item/L was prepared as follows: 100.0 mg test item were added to a 1 L volumetric flask containing test medium. C1: 6.25 Nominal test item concentration [mg/L] ; C2: 12.50 Nominal test item concentration [mg/L]; C3: 25.00 Nominal test item concentration [mg/L]; C4: 50.00 Nominal test item concentration [mg/L]; C5: 100.00 Nominal test item concentration [mg/L]

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Species: Daphnia magna
- code of the culture: Dm 29/02/20
- Age: < 24 hours old
- Diet: No feeding during the test
- Housing (before the test): Under breeding conditions, the test organisms were fed with algae suspension (Pseudokirchneriella subcapitata) and were maintained in reconstituted water.
- pH of medium: 6 - 9
- dissolved oxygen: ≥ 3 mg/L
- maintained in thermostatic chamber at the temperature range of 20±2°C, with a photoperiod of 16 hours light and 8 hours dark.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

between 140 and 250 mg/L (as CaCO3)

Test temperature:

in the range of 20.0 - 20.5 °C

pH:

in the range of 6-9

Dissolved oxygen:

≥ 3 mg/L

Nominal and measured concentrations:

Nominal test item concentration [mg/L]: 6.25 - 12.50 - 25.00 - 50.00 - 100.00 mg/L

Details on test conditions:

The test was conducted in a thermostatic chamber designed to maintain the test conditions specified in the protocol and in agreement with OECD 202 (2004). The temperature and light intensity values in the chamber were measured continuously during the test using a data-logger system.
During the exposure period, the temperature measured in the medium was in the range of 20.0 - 20.5 °C, the temperature measured in the thermostatic chamber was in the range of 19.4 - 20.3 °C, the light intensity was at maximum 862 Lux and a photoperiod of 16 hours light and 8 hours dark was applied.
Dissolved oxygen concentration and pH values were measured in the treated and untreated solutions, at the beginning and at the end of the test and at the test solutions renewal. The hardness of the medium was measured at the start of the test. The test solutions were not aerated and the daphnids have not been fed during the test.
During the test, the vessel position was modified daily and recorded in the study notebook; the vessels were positioned without a predetermined order, in order to minimise the influence of spatial differences of incubation conditions.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

54.93 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

76.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No other effects (e.g. behavioural effects, discoloration or trapping) were observed on the exposed organisms.

Reported statistics and error estimates:

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 nominal concentrations of test item.

Percent immobilisation after 24 and 48 hours of exposure of Daphnia magna to the test item:

Code	Nominal test item conc. [mg/L]	Number of exposed daphnids	Response at 24 h		Response at 48 h
			Number of immobilised	%I	Number of immobilised	%I
CTRL	0.00	20	0	0	0	0
CT SOLV	0.00	20	0	0	0	0
C1	6.25	20	0	0	0	0
C2	12.50	20	0	0	0	0
C3	25.00	20	0	0	0	0
C4	50.00	20	0	0	1	5
C5	100.00	20	7	35	17	85

According to OECD 202 (2004), the validity criteria were evaluated: Validity criteria results of the immobilisation test with Daphnia magna:

Validity criteria	Threshold	Observed
Immobilised daphnids in the untreated group, at the end of the test:	< 10%	0%
Dissolved oxygen concentration at the end of the test in treated and untreated groups:	≥ 3 mg/L	7.51 - 7.88 mg/L

Accordingly, all validity criteria are met.

Summary of the analytical results for fresh and aged media samples from the biological part of the study with Daphnia magna:

Code	Nominal conc. [mg test item/L]	Mean of measured conc. [mg test item/L]	Mean Recovery (%)
CTRL	0.00	< LOD *	n.d.
CT SOLV	0.00	< LOD *	n.d.
C1	6.15	5.56	90.44
C2	12.30	11.59	94.26
C3	24.59	21.98	89.38
C4	49.19	46.40	94.32
C5	98.37	91.71	93.23

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

n.d. = not determinable.

The analytical determination of the content of test item in the test solutions showed recovery values within the range 80-120% of the nominal values (with a mean recovery of 92.15% in the fresh samples and 92.51% in the aged samples), therefore the data evaluation was performed using the nominal concentrations.

Effect on Daphnia magna after 48 h-exposure to the Test item, in relation to the control:

Code	Test item concentration [mg/L]	%I
C1	6.25	0
C2	12.50	0
C3	25.00	0
C4	50.00	5
C5	100.00	85 +

+ indicates a significant difference compared to the untreated control (Step-down Cochran-Armitage Test Procedure (α = 0.050; one-sided greater

 

Effect concentrations as calculated by Probit analysis are presented in the following table.

 

Effect concentrations of the Test item on Daphnia magna after 48 h exposure:

Endpoint (48 hours)	Test item concentration [mg/L]	Confidence limits
		Lower	Upper
EC10	54.93	39.76	64.69
EC20	61.54	47.64	71.27
EC50	76.50	65.03	88.84
LOEC	100.00	-	-
NOEC	50.00	-	-

Endpoint (48 hours) Test item concentration [mg/L]   C.L.: Confidence Limit EC10 54.93 (95 % C.L. 39.76-64.69) EC20 61.54 (95 % C.L. 47.64-71.27) EC50 76.50 (95 % C.L. 65.03-88.84) LOEC 100.00 NOEC 50.00

Validity criteria fulfilled:

yes

Conclusions:

For 2-Amino-5-Chloro-N,3-Dimethylbenzamide the water hazard on Daphnia magna after 48 hours of exposure was determined with the following results:
- EC10 54.93 mg/L; EC20 61.54 mg/L; EC50 76.50 mg/L;
- Corresponding LOEC 100.00 mg/L;
- Corresponding NOEC 50.00 mg/L.
Additionally no other effects were observed on the exposed organisms.

No classification as hazardous according to EU regulation 1272/2008 (CLP) is required.

Executive summary:

The acute immobilisation test was performed, under semi-static conditions, to assess the effects of test item 2-Amino-5-Chloro-N,3-Dimethylbenzamide on Daphnia magna after 48 hours of exposure.

20 daphnids, less than 24 hours old, were exposed to 6.25 - 12.50 - 25.00 - 50.00 - 100.00 mg/L of test item in ISO medium containing 0.01 % of the solvent DMSO, plus two untreated control groups (with and without solvent).

The analytical determination of the active ingredient content showed a mean recovery of 92.15% in the fresh samples and 92.51% in the aged samples. Accordingly, the assessment of the effects was based on the nominal concentrations of the test item.

After 48 hours, the endpoints based on the test item nominal concentrations were estimated to be the following:

- EC_10:54.93 mg/L; EC_20:61.54 mg/L; EC_50:76.50 mg/L;

- Corresponding LOEC: 100.00 mg/L;

- Corresponding NOEC: 50.00 mg/L.

No other effects were observed on the exposed organisms.

The validity criteria of OECD 202 (2004) were fulfilled.

Description of key information

After 48 hours, the endpoints based on the test item nominal concentrations were estimated to be the following:

- EC_10:54.93 mg/L; EC_20:61.54 mg/L; EC_50:76.50 mg/L;

- Corresponding LOEC: 100.00 mg/L;

- Corresponding NOEC: 50.00 mg/L.

No other effects were observed on the exposed organisms.

The validity criteria of OECD 202 (2004) were fulfilled.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

76.5 mg/L