2'-chloroacetoacetanilide

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:

2010-03-09 to 2010-03-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted and reported according to the OECD Guideline for the testing of chemicals 202 and principles of GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2'-chloroacetoacetanilide

EC Number:

202-269-1

EC Name:

2'-chloroacetoacetanilide

Cas Number:

93-70-9

Molecular formula:

C10H10ClNO2

IUPAC Name:

2'-chloroacetoacetanilide

Details on test material:

- Molecular formula: C10H10ClNO2
- Molecular weight: 211.65 g/mol
- Physical state: Solid, white powder
- Expiration date of the lot/batch: 2010-08-01
- Storage condition of test material: Room temperature, protected from moisture and light

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No details.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and control in fresh (0 h) and old media (48 h)
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions.
- 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: A stock solution (200 mg/L was weighed out) was freshly prepared with dilution water. Dispersion treatment was agitation.
- Eluate: Dilution water
- Differential loading: 12.5 - 25.0 - 50.0 - 100 - 200 mg/L
- 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: 267 (mg CaCO3/L)

Test temperature:

21.0 °C

pH:

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

200 7.53
100 7.55
50 7.52
25 7.56
12.5 7.61
Control 7.66

Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)
Nominal
Test Item
Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
200 7.37 7.46 7.42 7.43
100 7.38 7.50 7.57 7.46
50 7.46 7.43 7.56 7.51
25 7.43 7.52 7.45 7.42
12.5 7.36 7.56 7.39 7.43
Control 7.64 7.60 7.49 7.46

Dissolved oxygen:

Water Quality Parameters at Test Start (0 h, Fresh Media)
(measured in one replicate per concentration level and control)
Nominal
Test Item
Concentration
[mg/L] Dissolved
O2-Concentration
[mg/L]
200 8.29
100 8.61
50 8.76
25 8.87
12.5 8.89
Control 8.29

Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)
Nominal
Test Item
Concentration
[mg/L] Dissolved O2 -Concentration [mg/L]
Replicates
1 2 3 4
200 8.20 8.17 8.01 8.10
100 8.19 8.10 8.37 7.34
50 8.13 8.11 8.17 8.16
25 8.20 8.20 8.00 7.93
12.5 8.40 8.42 8.40 8.39
Control 8.63 8.48 8.38 8.34

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

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
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
test item
concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 60 20 40 100 100 100
10 0 0 0 0 10 5
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.:

20.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: (19.4 - 22.0)

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: 1.44 (CI 1.35 - 1.54) 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

Immobilization Rates of the Definitive Testafter 24 and 48 h of Exposure

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

IMMOBILIZATION [%]
Acetessig-o-chloranilid TTR Nominal Test Item Concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
200	80	100	100	100	95	100	100	100	100	100
100	80	100	100	100	95	80	100	100	100	95
50	100	100	100	100	100	100	100	100	100	100
25	80	60	100	80	80	80	80	100	80	85
12.5	0	0	0	0	0	0	0	0	0	0
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 Acetessig-o-chloranilid TTR the 48-h EC50 for Daphnia magna was estimated to be 20.7 mg/L (95 % confidence limits: 19.4 – 22.0 mg/L).
The NOEC after 48 h was determined to be 12.5 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 h was determined to be 25.0 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:

The study was conducted and reported according to the OECD Guideline for the testing of chemicals 202 and principles of GLP.

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the test item

The study was conducted under static conditions over a period of 48 h with 5 nominal test item concentrations ranging from 12.5 to 200 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). Details of the analytical method are presented in part 10.
The recoveries of the test item Acetessig-o-chloranilid TTRwere determined to be in the range of 93 to 98 % of the nominal values in the fresh media (0 h) and old media (48 h) in all concentration levels, demonstrating that the concentration of Acetessig-o-chloranilid TTR 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 Acetessig-o-chloranilid TTR. The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0 and 48 h, 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 Acetessig-o-chloranilid TTR)

	Test Duration [h]	Nominal Concentration of the Test Item Acetessig-o-chloranilid TTR [mg/L]	Confidence Interval p = 95% [mg/L]
EC10	24	19.6	---
	48	16.5	---
EC50	24	22.6	21.4 – 23.8
	48	20.7	19.4 – 22.0
EC100	24	50.0	---
	48	50.0	---
NOEC	48	12.5	---
LOEC	48	25.0	---