1-chloro-2,5-dimethoxy-4-nitrobenzene

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:

20110-06-02 to 2010-08-04

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:

1-chloro-2,5-dimethoxy-4-nitrobenzene

EC Number:

230-086-7

EC Name:

1-chloro-2,5-dimethoxy-4-nitrobenzene

Cas Number:

6940-53-0

Molecular formula:

C8H8ClNO4

IUPAC Name:

1-chloro-2,5-dimethoxy-4-nitrobenzene

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)
- Method: OECD 23, WAF
- Eluate: filtered through 0.45 µm sieve
- 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:

yes

Total exposure duration:

48 h

Post exposure observation period:

no

Test conditions

Hardness:

9°dH (160.63 mg/L as CaCO3) at test initiaition

Test temperature:

21.1°C +/- 0.5°C

pH:

8.25 +/- 0.29

Dissolved oxygen:

99 +- 1.8 % (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.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: measured: 13 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 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)

No EC50 value could be derived since no mortality was performed up to the tested concentration of 100 mg/L (nominal loading). Hence, the NOEC is 100 mg/L (nominal loading).

For nominal 100 mg/L the corresconding measured value from the chemical analysis was found to be 13 mg/L.

Raw data 48 h

	Control	0.01	0.1	1	10	100
		[mg/L]
	immobilised daphnids after 48 h
Group 1	0	0	0	0	0	0
Group 2	0	0	0	0	0	0
Group 3	0	0	0	0	0	0
Group 4	0	0	0	0	0	0
S	0	0	0	0	0	0
%	0	0	0	0	0	0

Toxic Reference

	24 h			48 h
K2Cr2O7	1.0	2.0		1.0		2.0
[mg/L]	immobilised daphnids
Group 1	0		0	0	4
Group 2	0		0	0	5
Group 3	1		2	1	5
Group 4	0		0	0	5
S	1		2	1	19
%	5		10	5	95

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.

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 of 1-Chloro-2,5-dimethoxy-4-nitrobenzene in the samples during the test. Samples were taken after initiation of the test, after 24 hours and 48 hours at all concentration levels. Control and 100 mg/L were analysed at t = 0h,        t = 24 h and t = 48 h.

Test item	Sampling (h)	1-Chloro-2,5-dimethoxy-4-nitrobenzene
nominal (mg/L)		mg/L
0	0 fresh	< LOQ
	24 aged	< LOQ
	48 aged	< LOQ
100	0 fresh	13
	24 aged	13
	48 aged	12

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 from 2.00 mg/L to 0.05 mg/L with r²> 0.995

Nominal concentration (ng/mL)	Peak area	Calculated concentration (ng/mL)
2000	775 113	2041.006
1000	340 618	944.212
500	141 016	440.363
200	41 602	189.409
100	17 772	129.253
50	8 464	105.757

% O₂saturation of the test solutions

Test item	nominal test item concentration [mg/L]
	Control	0.01	0.1	1	10	100
Time [h]	Oxygen
0 fresh	96	96	97	96	96	97
24 aged	99	100	100	101	100	101
48 aged	100	99	99	100	100	100
Mean	98.3	98.3	98.7	99.0	98.7	99.3
Std. dev.	2.1	2.1	1.5	2.6	2.3	2.1
Mean	99
Std. dev.	1.8

pH-values of the test solutions

	nominal test item concentration [mg/L]
	Control	0.01	0.1	1	10	100
Time [h]	pH
0 fresh	7.87	7.87	7.85	7.82	7.88	7.86
24 aged	8.42	8.45	8.45	8.47	8.49	8.49
48 aged	8.36	8.41	8.44	8.45	8.46	8.46
Mean	8.22	8.24	8.25	8.25	8.28	8.27
Std. dev.	0.30	0.32	0.34	0.37	0.34	0.36
Mean	8.25
Std. dev.	0.29

Temperature of the test solutions

	nominal test item concentration [mg/L]
	Control	0.01	0.1	1	10	100
Time [h]	Temperature
0 fresh	21.8	21.8	21.9	21.9	21.9	21.6
24 aged	20.4	20.4	20.8	21.1	20.9	20.9
48 aged	20.6	20.7	20.7	21.0	20.9	20.9
Mean	20.9	21.0	21.1	21.3	21.2	21.1
Std. dev.	0.8	0.7	0.7	0.5	0.6	0.4
Mean	21.1
Std. dev.	0.5

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity study (48h) on daphnia was determined . Up to the highest test concentration of 100 mg/L (nominal loiading) no mortalitity could be observed.

Executive summary:

In a valid GLP study following Guideline EC-method C.2 and OECD 202 (2004) the short term toxicity of 1-Chloro-2,5-dimethoxy-4-nitrobenzene to aquatic invertebrates, represented by Daphnia magma was determined.

According to the results of the test, theEC₅₀(48 h)of the test item was determined to be> 100mg/L(nominal).The correspondingNOEC (48 h)was100 mg/L(nominal).The correspondingactualvalueswere >13 mg/Lfor theEC₅₀(48 h)and 13mg/Lfor theNOEC (48 h).