4-chloro-2,5-dimethoxyaniline

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-20 to 2010-07-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, followed international guidelines, no deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-2,5-dimethoxyaniline

EC Number:

228-782-0

EC Name:

4-chloro-2,5-dimethoxyaniline

Cas Number:

6358-64-1

Molecular formula:

C8H10ClNO2

IUPAC Name:

4-chloro-2,5-dimethoxyaniline

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations and blank
- Sampling method: from the middle of the water column and from the middle of the tanks
- Sample storage conditions before analysis: frozen, thawed just befor analysing

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Differential loading: by dilution
- Controls: yes, blank controls
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: Clone V
- Source: Laboratory bred, originally purchased in a healthy condition from the Umweltbundesamt in Berlin/Germany.
- Age at study initiation (mean and range, SD): freshly hatched, age between 6 - 24 hours
- Peripheral shell growth removed prior to test initiation: no
- Method of breeding: single culture (one daphnid per 100 mL) in water composed of dechlorinated drinking water and deionised water.
- Feeding during test: no
- Food type: not applicable
- Amount: not applicable
- Frequency: not applicable

ACCLIMATION
- Acclimation period: not applicable, breeding water is the same as test water
- Acclimation conditions (same as test or not): yes
- Type and amount of food: not during test
- Feeding frequency: not during test
- Health during acclimation (any mortality observed): not applicable

QUARANTINE (wild caught)
- Duration: not applicable
- Health/mortality: not applicable

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

no

Test conditions

Hardness:

11°dH

Test temperature:

20.3 +- 0.4 °C (Std. Dev.)

pH:

8.31 +- 0.2 (Std. Dev.)

Dissolved oxygen:

100 +- 1.1 % (Std. Dev.)

Salinity:

no data

Nominal and measured concentrations:

see block any other information on material and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: 100 ml beaker, filled 50 ml
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): 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): not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated drinking water and deionised water
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: "dechlorinated"
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: test was performed in the dark since the test item is known to be substject to photolysis
- Light intensity: not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : swimming behaviour

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2,2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes
- Test concentrations:: 0-0,01 - 0,1 - 1-10-10 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

3.125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit: 5.27 - 7.95 [mg/L]

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: not reported
- Observations on body length and weight: not reported
- Other biological observations: not reported
- Mortality of control: not signifcant
- Other adverse effects control: not reported
- Abnormal responses: not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: yes

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality: see table in block "any other information on results..."
- EC50/LC50: see table in block "any other information on results..."
- Other:

Any other information on results incl. tables

I. EC_x-values of daphnids exposed to the test item (nominal / actual concentrations)

	4-Chlor-2,5-dimethoxyanilin[mg/L] (nominal)
	24 h	48h
NOEC	3.125	3.125
EC100	> 50.0	> 50.0
EC50	9.47	6.47
95 % confidence limit of EC50	7.36 – 12.19	5.27 – 7.95

Raw data 48 h

	Control	1.56	3.125	6.25	12.5	25.0	50.0
		mg/L
	immobilised daphnids after 48h
Group 1	0	0	0	3	5	4	5
Group 2	0	0	0	3	5	5	5
Group 3	0	0	0	2	5	5	5
Group 4	0	0	1	3	4	5	4
S	0	0	1	11	19	19	19
%	0	0	5	55	95	95	95

Toxic Reference

	24 h			48 h
K2Cr2O7	1.0	2.0		1.0		2.0
[mg/L]	immobilised daphnids
Group 1	0		4	0	5
Group 2	0		4	1	5
Group 3	0		3	0	5
Group 4	1		5	1	5
S	1		16	2	20
%	5		80	10	100

The results indicate an EC₅₀(48 h) of the reference item potassium dichromate between 1.0 mg/L and 2.0 mg/L. Since the results fall within the historical data generated with the reference item at the testing facility, the daphnids were suitable for the determination of the toxicological effects of the test item.

II. Analytical results

The test was performed under static conditions. The analytical verification of test item concentrations in daphnid test medium was done by analysing the content of4-Chlor-2,5-dimethoxyanilinin the samples during the test. Samples were analysed at t = 0 h and t = 48 h from all concentration levels.

Test item	Sampling (h)	4-Chlor-2,5-dimethoxyanilin
nominal (mg/L)		mg/L	% of nominal	% mean
0	0	n. d.	-	-
	48	n. d.	-
1.56	0	1.12	72	74
	48	1.19	76
3.125	0	2.301	74	79
	48	2.602	83
6.25	0	4.59	73	80
	48	5.38	86
12.5	0	8.38	67	77
	48	10.71	86
25.0	0	16.5	66	74
	48	20.5	82
50.0	0	30.0	60	76
	48	45.6	91

LOQ: 0.4 mg/L test item

The test was performed within the solubility limit of 1-Chloro-2,5-dimethoxy-4-nitrobenzene. At the highest test concentration of 100 mg/L nominal only  13 mg 1-Chloro-2,5-dimethoxy-4-nitrobenzene/L was found after a 48-hours stirring period and filtration. The toxicological endpoints were evaluated using nominal and actual concentrations of the test item.

Recovery of 1-chloro-2,5-dimethoxy-4-nitrobenzene from the test item spiked into test medium

The recovery was determined by fortification of test medium with the test item at the concentration levels

Fortification level (mg/L)	Found (mg/L)	Recovery (%)	Mean Recovery ±RSD (%)
0.400	0.400 0.441 0.441 0.443 0.447	100 110 110 111 112	109±4
100	112 103 105 108 107 89 83	112 103 105 108 107 89 83	101±11

Mean recoveries and relative standard deviations per fortification fulfil the criteria of guideline SANCO/3029/99 (70-110 % mean recovery, ≤ 20 % RSD).

Linearity

The detector response for HPLC-MS/MS analysis was linear within the range from1.00 mg/L to 0.02 mg/Lwith r²> 0.999

Typical calibration data for analysis of4-Chloro-2,5-dimethoxyanilineby HPLC-MS/MS (quantifier ion transition m/z 188®172.9)

Nominal concentration (ng/mL)	Peak area	Calculated concentration (ng/mL)
1000	1 460 409	997.708
500	736 748	503.551
200	297 874	204.160
100	142 533	98.124
50	69 223	48.083
20	25 669	18.373

% O₂saturation of the test solutions

	nominal test item concentration [mg/L]
	Control	1.56	3.125	6.25	12.5	25.0	50.0
Time [h]	Oxygen
0	103	102	99	98	99	101	100
24 aged	101	100	100	100	100	101	101
48 aged	100	101	100	100	99	100	100
Mean	101.3	101.0	99.7	99.3	99.3	100.7	100.3
Std. dev.	1.5	1.0	0.6	1.2	0.6	0.6	0.6
Mean	100
Std. dev.	1.1

pH-values of the test solutions

	nominal test item concentration [mg/L]
	Control	1.56	3.125	6.25	12.5	25.0	50.0
Time [h]	pH
0	8.12	7.97	7.88	8.17	8.06	8.08	8.09
24 aged	8.34	8.35	8.39	8.44	8.43	8.43	8.46
48 aged	8.45	8.46	8.48	8.51	8.49	8.49	8.52
Mean	8.30	8.26	8.25	8.37	8.33	8.33	8.36
Std. dev.	0.17	0.26	0.32	0.18	0.23	0.22	0.23
Mean	8.31
Std. dev.	0.2

Temperature of the test solutions

	nominal test item concentration [mg/L]
	Control	1.56	3.125	6.25	12.5	25.0	50.0
Time [h]	Temperature
0	20.8	20.1	20.8	20.6	20.6	20.7	20.9
24 aged	20.3	20.4	20.4	20.5	20.3	20.4	20.5
48 aged	19.7	19.8	19.9	19.9	19.7	19.6	19.9
Mean	20.3	20.1	20.4	20.3	20.2	20.2	20.4
Std. dev.	0.6	0.3	0.5	0.4	0.5	0.6	0.5
Mean	20.3
Std. dev.	0.4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity study (48h) on daphnia was determined . The EC50 was determined to be 6.47 mg/L.

Executive summary:

In a valid GLP study following Guideline EC-method C.2 and OECD 202 (2004) the short term toxicity of 4-Chlor-2,5-dimethoxyanilin to aquatic invertebrates, represented by Daphnia magma was determined.

According to the results of the test, the EC₅₀ (48 h) of the test item was determined to be 6.47 mg/L (nominal). The corresponding NOEC (48 h) was 3.125 mg/L (nominal).