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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 concentrationsopen allclose all
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
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. ECx-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).