2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

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:

2012-08-20 to 2012-08-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Concentrations: Saturated solution as limit concentration
- Sampling method: No analytical monitoring

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The saturated solution (1mg/L was weighed out) was prepared with dilution water two days prior to application. The saturated solution was stirred with approximately 1100 rpm for 48 hours at room temperature. Undissolved particles were removed by centrifugation with 10000 rpm for 10 minutes
- Eluate: Dilution water
- Differential loading: None, limit test
- Controls: Four replicates of dilution water without test item tested under the same conditions as the test groups.

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

DILUTION WATER:
ISO test water, according to OECD 202, Annex 3:
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2. 2 H2O 294
MgSO4 . 7 H2O 123
pH-value: 7.8 +/- 0.2

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

Dilution water at test start: 171 (mg CaCO3/L)

Test temperature:

21.7 °C

pH:

Water Quality Parameters at Test Start (0 h)
(measured in one additional replicate of the saturated solution and the control)
Test Item
Nominal Test Item Concentration
[mg/L] pH-Value
1.00
(Saturated solution) 7.63
Control 7.56

Water Quality Parameters at the Test End (48 h)
(measured in all replicates)
Test Item
Nominal Test Item Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
1.00
(Saturated solution) 7.63 7.68 7.69 7.73
Control 7.46 7.66 7.64 7.65

Dissolved oxygen:

Water Quality Parameters at Test Start (0 h)
(measured in one additional replicate of the saturated solution and the control)
Test Item
Nominal Test Item Concentration
[mg/L] Dissolved O2-Concentration [mg/L]
1.00
(Saturated solution) 7.99
Control 8.42

Water Quality Parameters at the Test End (48 h) (measured in all replicates)
Test Item
Nominal Test Item Concentration
[mg/L] Dissolved O2 –Concentration [mg/L]
Replicates
1 2 3 4
1.00
(Saturated solution) 8.08 8.28 8.14 8.32
Control 7.96 7.71 7.90 7.36

Salinity:

Not measured, 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: see above
- Conductivity: Dilution water at test start: 960 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) :
Immobility was determined in all groups after 24 h and 48 h. An animal was considered to be immobile, if it was not able to swim within 15 sec after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Range finding study: Yes
Immobilization Rates in the Preliminary Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Test Item
IMMOBILIZATION [%]
Nominal Test Item Concentration

[mg/L] 24 hours 48 hours
Replicates Replicates
1 2 MV 1 2 MV
1.00
(Saturated solution) 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, no suitable method for determination of the test item could be established.

Duration:

48 h

Dose descriptor:

other: EC10/50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, no suitable method for determination of the test item could be established.

Details on results:

- Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.78 (CI 1.51 - 2.06) mg/L

Reported statistics and error estimates:

The effects of the saturated solution of the test item were deduced directly from the observed immobilization rates.

EC50-value was only calculated for the reference item by sigmoidal dose-response regression. The confidence interval for EC50 was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5

The percentage immobility, determined in the saturated solution with 1.00 mg/L of the test itemand the control after 24 and 48 h under static conditions, is presented in the table above. There was no effect observed, neither in the saturated solution nor in the control group

Immobilization Rates of the Definitive Test after 24 and 48 hours of Exposure

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

Test Item	IMMOBILIZATION [%]
Nominal Test Item Concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
1.00 (Saturated solution)	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

At the saturated solution* with nominal 1.00 mg/L of the test item, no effects were observed on Daphnia magna.
(*) Saturated solution: The maxium dissolved concentration of the test item that chan be achieved under the test conditions in the test medium according to OECD Series No. 23 (2000).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the saturated solution* with 1.00 mg/L of the test item were determined according to OECD 202 (2004) and compliant to GLP.

The limit test was conducted under static conditions over a period of 48 hours.

Twenty daphnids were exposed to the limit concentration and the control. No analytical monitoring was carried out, because no suitable method for the determination of the hardly soluble test item could be established.

All effect values given are based on the nominal concentration of the test item.

Water quality parameters (pH-value and dissolved oxygen concentration), measured in the fresh (0 and 24 h) and old media (24 and 48 h), were determined to be within the acceptable limits. The validity criteria of the test guidelines were fulfilled.

EC-Values, NOEC and LOEC

(based on the nominal concentration of the test item)

Test Item
	Test Duration [h]	Nominal Concentration of the Test Item [mg/L]	Confidence Interval p = 95% [mg/L]
EC10 / EC50 / EC100	24	> 1.00	not applicable
	48	> 1.00	not applicable
NOEC	48	1.00	---
LOEC	48	> 1.00	---

(*) Saturated solution: The maxium dissolved concentration of the test item that chan be achieved under the test conditions in the test medium according to OECD Series No. 23 (2000).

Description of key information

At the saturated solution* with nominal 1.00 mg/L of the test item, no effects were observed on Daphnia magna.
(*) Saturated solution: The maxium dissolved concentration of the test item that chan be achieved under the test conditions in the test medium according to OECD Series No. 23 (2000).