4-nitro-o-anisidine

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:

2009-11-10 to 2009-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to current OECD guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-nitro-o-anisidine

EC Number:

202-588-6

EC Name:

4-nitro-o-anisidine

Cas Number:

97-52-9

Molecular formula:

C7H8N2O3

IUPAC Name:

2-methoxy-4-nitroaniline

Constituent 2

Reference substance name:

PNOA

IUPAC Name:

PNOA

Details on test material:

- Name of test material (as cited in study report): p-Nitro-o-anisidin
- Molecular formula: C7H8N2O3
- Molecular weight: 168.15 g/mol
- Physical state: Solid, yellow powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and control were analytically verified via HPLC after 0 h (freh media) and 48 h (old media).
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions. The analytical method was validated according to SANCO 3029/99 rev.4 (11/07/00).
- Sample storage conditions before analysis:All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution of 80.0 mg/L was freshly prepared with dilution water. The stock solution was treated with ultrasound for 30 min and stirred with approximately 1100 rpm for 1 hour at room temperature.
- Eluate: Dilution water
- Differential loading: 2.50 - 5.00 - 10.0 - 20.0 - 40.0 - 80.0 mg/L (dilution factor 2)
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

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:

Dilution water at test start: 263 (mg CaCO3/L)

Test temperature:

20.5 °C

pH:

Water Quality Parameters at Test Start (0 hours, Fresh Media)
(measured in one replicate per concentration level and control)
Nominal
Test Item
Concentration
[mg/L] pH-Value



80.0 7.49
40.0 7.51
20.0 7.51
10.0 7.49
5.00 7.49
2.50 7.48
Control 7.98

Water Quality Parameters at Test End (48 hours, Old Media)
(measured in all replicates)
Nominal
Test Item
Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
80.0 7.49 7.50 7.46 7.45
40.0 7.49 7.48 7.50 7.49
20.0 7.48 7.47 7.48 7.43
10.0 7.51 7.50 7.50 7.49
5.00 7.50 7.47 7.50 7.51
2.50 7.44 7.50 7.50 7.49
Control 7.47 7.51 7.51 7.53

Dissolved oxygen:

Water Quality Parameters at Test Start (0 hours, Fresh Media)
(measured in one replicate per concentration level and control)
Nominal
Test Item
Concentration
[mg/L] Dissolved
O2-Concentration

[mg/L]
80.0 7.40
40.0 7.96
20.0 8.20
10.0 8.14
5.00 8.30
2.50 8.21
Control 8.37

Water Quality Parameters at Test End (48 hours, Old Media)
(measured in all replicates)
Nominal
Test Item
Concentration
[mg/L] Dissolved O2 -Concentration [mg/L]
Replicates
1 2 3 4
80.0 6.93 6.92 6.80 7.23
40.0 6.92 6.91 7.04 6.78
20.0 6.97 6.78 6.89 7.07
10.0 7.36 7.05 7.20 7.34
5.00 7.38 7.15 7.23 7.31
2.50 7.07 7.25 7.26 7.20
Control 7.32 7.27 7.49 7.47

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- 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): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Acc. to Directive 92/69/EEC Method C.2, Annex 1
- Conductivity: Dilution water at test start: 640 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
Immobilization Rates of the Preliminary Test in [%]
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
test item
concentration
[mg/L] IMMOBILIZATION [%]
24 hours 48 hours
Replicates Replicates
1 2 MV 1 2 MV
100 100 100 100 100 100 100
10 10 20 15 10 20 15
1 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (6.20 - 8.07)

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.67 (CI 1.63 - 1.71) mg/L

Reported statistics and error estimates:

The NOEC, LOEC and the EC100-values were evaluated directly from the observation data without mathematical calculation.
EC10- and the EC50-values were calculated by sigmoidal dose-response regression. The confidence intervals for the EC50-values were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. The concentration effect relationships are shown graphically.

Any other information on results incl. tables

Percentage of Daphnids Incapable of Swimming after 24 and 48 h of Exposure

                 (n = 20, divided into 4 replicates with 5 daphnids each)

IMMOBILIZATION [%]
p-Nitro-o-anisidin Nominal Test Item Concentration [mg/L]	24 hours					48 hours
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
80.0	100	100	100	100	100	100	100	100	100	100
40.0	80	100	80	100	90	100	100	100	100	100
20.0	40	100	60	100	75	100	100	80	100	95
10.0	40	80	60	40	55	60	80	80	60	70
5.00	40	20	0	20	20	40	40	20	20	30
2.50	20	0	0	0	5	20	0	0	0	5
Control	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item p-Nitro-o-anisidin the 48-hour EC50 for Daphnia magna was estimated to be 7.07 mg/L (95 % confidence limits: 6.20 - 8.07 mg/L).
The NOEC after 48 hours was determined to be 2.50 mg/L as the highest tested concentration with no biologically significant effect (Immobilization rates of < 10 % are seen to be not significant according to the guideline). The LOEC after 48 hours was determined to be 5.00 mg/L as the lowest tested concentration with a biologically significant effect (Immobilization rates of > 10 % are seen to be significant according to the guideline).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the test item  p-Nitro-o-anisidin were determined according to OECD 202 (2004).

The study was conducted under static conditions over a period of 48 hours with 6 nominal test item concentrations ranging from 2.50 to 80.0 mg/L set up in a geometric series with a dilution factor of 2. 20 test organisms were exposed to each test concentration and control. All test concentrations and control were analytically verified via HPLC after 0 h (fresh media) and 48 h (old media).
The recoveries of the test item p-Nitro-o-anisidinat the biologically relevant concentration range of 2.50 to 20.0 mg/L were determined to be in the range of 98 to 102 % of the nominal values in the fresh media (0 hours) and old media (48 hours) in all concentration levels, demonstrating that the concentration of p-Nitro-o-anisidin was successfully maintained for the duration of the test.
All effect values, presented in Table 1, are based on the nominal concentrations of the test item p-Nitro-o-anisidin. The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0 and 48 hours, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1:    EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀ - Values, NOEC and LOEC

              (based on the nominal concentrations of the test item p-Nitro-o-anisidin)

	Test Duration [h]	Nominal Concentration of the Test Item p-Nitro-o-anisidin [mg/L]	Confidence Interval p = 95% [mg/L]
EC10	24	2.96	---
	48	3.04	---
EC50	24	9.91	7.32 – 13.4
	48	7.07	6.20 – 8.07
EC100	24	80.0	---
	48	40.0	---
NOEC	48	2.50	---
LOEC	48	5.00	---