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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:

25-27 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP.

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Name: NO.148 AQN
Lot No.: 00080
CAS No.: 214115-79-4
Chemical name: 4'-{[3-(diethylamino)propyl]carbamoyl}-9,10-dihydro-9,10-dioxoanthracene-2-carboxanilide
Purity: 96.81 %
Description: Pale yellow powder
Production date: 05 April 2010
Expiry date: 04 April 2012
Storage: room temperature (15 – 25 °C)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Concentrations of the test material in the test solution was determined at the beginning and at the end of the study.
At the start of the test five samples was taken from the test solution. At the end of the test four samples were taken from each of the test vessels. Both occasions one sample was taken from the control solution.
- Sample storage conditions before analysis: room temperature (15 – 25 °C)

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:Because the test item is poorly soluble in water, the stock solution used in the test was prepared by a method described below (see Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.):
A stock solution (nominally 100 mg/L) was prepared by mechanical dispersion one day before the start of the test. This solution was shaken for about 24 hours. After shaking the non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100 % v/v saturated solution.
- Controls: yes, the dilution water (ISO-medium) was used without of addition of the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna
- Source: Supplied by National Institute of Public Health.
- Age at study initiation (mean and range, SD): They were less than 24 h old at the beginning of the test.
- Method of breeding: Cultured under standardised conditions at the Ecotoxicological Laboratory of LAB Research Ltd.
- Feeding during test
- Food type: Before the test the Daphnia culture was fed with concentrated algal suspension of Pseudokirchneriella subcapitata. The test animals were not fed during the test.

ACCLIMATION
- Acclimation period: There was no acclimatization because the water used was similar to the culture water.
- Type and amount of food: Before the test the Daphnia culture was fed with concentrated algal suspension of Pseudokirchneriella subcapitata.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

None

Test conditions

Hardness:

The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Test temperature:

The water temperature was measured at the start and 24-hour intervals thereafter in each test vessel. The test temperature was in the range of 20.9 – 21.1 °C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 20.7 and 21.4 °C.

pH:

The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the test in each test vessel and was in the range of 7.68 – 7.84.

Dissolved oxygen:

The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the test and was in the range of 6.7 – 6.8 mg/L.

Salinity:

n/a

Nominal and measured concentrations:

Because a significant toxic response was not observed during the preliminary concentration range-finding test, only one test concentration at the solubility level of the test item in the test medium (100 % v/v saturated solution) and one control group was tested in a limit test. The corresponding measured geometric mean test item concentration was 0.95 mg/L.
The test results are based on the measured test item concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker (~50 mL)
- Material, size, headspace, fill volume: at least 4 mL test solution/animal
- Renewal rate of test solution (frequency/flow rate): without water renewal
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.
Separate stock solutions of individual trace elements were first prepared in deionised water. The medium was prepared of these different stock solutions, so it contained all trace elements (combined solution):

Stock solutions (single substance) Amount added to deionised water Stock solutions added to deionised water to prepare media
(g/L) (ml/L)
CaCl2 × 2 H2O 11.76 25
MgSO4×7 H2O 4.93 25
KCl 0.23 25
NaHCO3 2.59 25

- Intervals of water quality measurement: The temperature was measured at 0, 24 & 48 hours. The pH and dissolved oxygen was measured at 0 & 48 hours.

OTHER TEST CONDITIONS
- Photoperiod: The test was carried out in 16-hour light and 8-hour dark cycle.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Untreated control, 0.1, 1, 10, 50 & 100
- Results used to determine the conditions for the definitive study: Because a significant toxic response was not observed during the preliminary concentration range-finding test, only one test concentration at the solubility level of the test item in the test medium (100 % v/v saturated solution) and one control group was tested in a limit test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.95 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.95 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: There was no immobilisation in 20 daphnids exposed to each group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.
- Effect concentrations exceeding solubility of substance in test medium: yes

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: 1.77 mg/L, (95 % confidence limits: 1.46 – 2.15 mg/L)

Reported statistics and error estimates:

No statistical analysis was necessary because the lack of toxic effects.
The EC50, NOEC and LOEC were determined directly from the raw data.

Any other information on results incl. tables

VALIDITY

There were no immobilized animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

CONCENTRATIONS OF THE TEST ITEM

The measured concentration of the 100 % saturated solution was 1.03 mg/L at the start of the test and 0.88 mg/L at the end of the experiment. The calculated geometric mean concentration was 0.95 mg/L.

IMMOBILISATION

The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour (Table 2).

There was no immobilisation in 20 daphnids exposed to each group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Acute toxicity of the test material was assessed with acute immobilisation test on daphnia (Daphnia magna), over an exposure period of 48 hours in a static system.

Under the conditions of this Daphnia magna acute immobilisation study, the observed endpoints for the effect of the test material were the followings:

The 24h EC50 value: >0.95 mg/L

The 48h EC50 value: >0.95 mg/L

The 48h NOEC value: 0.95 mg/L

The 48h LOEC value: >0.95 mg/L

All validity criteria were met during the study.

Table 2: Number and percentage of immobilised animals

Test group	Number of treated animals	number of immobilised animals
		24 hours		48 hours
		number	percent	number	percent
Control	20	0	0	0	0
100% saturated solution (0.95 mg/L measured	20	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this Daphnia magna acute immobilisation study, the observed endpoints for the effect of the test material were the followings:
The 24h EC50 value: >0.95 mg/L
The 48h EC50 value: >0.95 mg/L
The 48h NOEC value: 0.95 mg/L
The 48h LOEC value: >0.95 mg/L
All validity criteria were met during the study.

Executive summary:

Acute toxicity of the test material was assessed with acute immobilisation test on daphnia (Daphnia magna), over an exposure period of 48 hours in a static system.

Because a significant toxic response was not observed during the preliminary concentration range-finding test, only one test concentration at the solubility level of the test item in the test medium (100 % saturated solution) and one control group were tested in a limit test. The corresponding measured geometric mean concentration was 0.95 mg/L.

The biologically results are based on the measured test item concentration.

The test design included four replicates at each test group (test item and controls). Each group comprised twenty Daphnia, five in each of the four replicate vessels, each containing approximately 40 mL test dilution.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study, the observed endpoints for the effect of NO.148 AQN were the followings:

The 24h EC50 value: >0.95 mg/L

The 48h EC50 value: >0.95 mg/L

The 48h NOEC value: 0.95 mg/L

The 48h LOEC value: >0.95 mg/L