Registration Dossier

Ecotoxicological information

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:
11 March 2008 and 19 August 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Test samples were analysed either directly or diluted with water* to give final theoretical concentrations of 1.0 to 11 mg/l as test material.

Standard solutions of diethylamine were prepared in test medium at a nominal concentration of 5.0 mg/l.

* Prepared by ELGA Purelab Option R-15BP water purification

- Sampling method: The concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of a single peak.

The method was developed by the Department of Analytical Services, Safepharm Laboratories Limited.

- Sample storage conditions before analysis: Room temperature over silica gel under nitrogen in the dark

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test the test material was dissolved directly in reconstituted water.

An amount of test material (200 mg) was dissolved in reconstituted water and the volume adjusted to 2 litres to give the 100 mg/l test concentration. Aliquots (5.0, 9.0, 16, 28, 50, 90, 160 and 280 ml) of the 100 mg/l test concentration were each separately dispersed in a final volume of 500 ml of reconstituted water to give the 1.0, 1.8, 3.2, 5.6, 10, 18, 32 and 56 mg/l test concentrations respectively.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Information provided by the Sponsor indicated that the test material rapidly degrades to diethylamine, ethanol and a water soluble silicon compound. The concentration and stability of the degradation product diethylamine in the test preparations were verified by chemical analysis at 0 and 48 hours (see attachment 1).

- Eluate: Not applicable

- Controls: A positive control (Safepharm Laboratories Project No: 0039/0977) conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Exposure conditions for the positive control were similar to those used in the definitive test.

The temperature was maintained at 21°C.

- Chemical name of vehicle : Not applicable

- Concentration of vehicle in test medium: Not applicable

- Evidence of undissolved material : None


Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia Magna

- Source: Derived from in-house laboratory cultures.

- Age at study initiation: The test was carried out using 1st instar Daphnia Magna.

- Feeding during test: Received no food during exposure

ACCLIMATION
- Acclimation period: Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

These young were removed from the cultures and used for testing.

- Acclimation conditions: Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 degC.

The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

- Type and amount of food:Each culture was fed daily with a suspension of algae (Chlorella sp.). Culture conditions ensured that reproduction was by parthenogenesis.

The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

- Health during acclimation: No mortality observed


Test Water:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

The reconstituted water is defined in Attachment 2.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not recorded.

Test conditions

Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
Temperature was maintained at 21 deg C to 22 deg C throughout the test.

Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
Dissolved oxygen:
Aerated until the dissolved oxygen concentration was approximately air-saturation value.
Salinity:
Not recorded.
Nominal and measured concentrations:
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1.

Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
Details on test conditions:
TEST SYSTEM
As in the range-finding tests 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the study 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21 deg C to 22 deg C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test material.

The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water with a conductivity of <5 µS cm-1.

- Total organic carbon: Not recorded

- Particulate matter: Not recorded

- Metals: Not recorded

- Pesticides: Not recorded

- Chlorine: Not recorded

- Alkalinity: pH of 7.8 ± 0.2

- Ca/mg ratio: The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

- Conductivity: Not recorded

- Culture medium different from test medium: The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

- Intervals of water quality measurement: Not recorded

Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted (if necessary) with NaOH or HCl.

- Photoperiod: Photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

- Light intensity: Not recorded


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Water samples were taken from the control (replicates R1 – R2 pooled) and each test group (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis.

Duplicate samples were taken and stored at approximately -20 deg C for further analysis if necessary.

The method of analysis, stability, recovery and test preparation analyses for the degradation product diethylamine are described in Attachment 1.

- Justification for using less concentrations than requested by guideline: Not applicable

- Range finding study
- Test concentrations: The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l. The test material was dissolved directly in reconstituted water.

An amount of test material (50 mg) was dissolved in reconstituted water and the volume adjusted to 500 ml to give the 100 mg/l test concentration from which serial dilutions were prepared in reconstituted water to give the remainder of the test concentrations of 10, 1.0 and 0.10 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20 deg C in the dark. The test was conducted using semi-static test conditions. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.

The range-finding test was conducted in the dark using semi-static test conditions as it was conducted prior to the stability analysis which showed the degradation product diethylamine to be stable in the test medium in the light over a 48-Hour period.

The control group was maintained under identical conditions but not exposed to the test material.

- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
88 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL of 64 - 160 mg/l. NOEC = 10mg/l
Details on results:
- Behavioural abnormalities: Not recorded

- Other biological observations: None recorded

- Immobilisation of control:
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Safepharm Laboratories Project No: 0039/0977) during the positive control are given in the following table.

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 24 hours and the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)
3 > 3.2* -
24 0.97 0.85 - 1.1
48 0.7 0.62 - 0.8


The No Observed Effect Concentration after 24 and 48 hours were 0.56 and 0.32 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slope and standard error of the response curve at 48 hours was 11 (SE = 2.3). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of the response curve at 24 hours.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.78 mg/l (sd = 0.22).

- Other adverse effects control: No other effects observed.

- Abnormal responses: None recorded

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None recorded

- Effect concentrations exceeding solubility of substance in test medium:
Results with reference substance (positive control):
- Results with reference substance
Information provided by the Sponsor indicated that the test material rapidly degrades to diethylamine, ethanol and a water soluble silicon compound.
The test concentrations were monitored by analysis of the test samples for diethylamine only.

Analysis of the test preparations at 0 hours (see Attachment 1) showed measured concentrations of diethylamine to range from 81% to 116% of nominal value with the exception of the 5.6 and 100 mg/l test concentrations which showed measured concentrations of 143% and 78% respectively. Given that the overall mean measured concentration at 0 hours was 97% of nominal it was considered that these values did not affect the outcome of the study.

Analysis of the test preparations at 48 hours showed measured concentrations of diethylamine to range from 81% to 103% of nominal value with the exception of the 1.8, 5.6, 10 and 32 mg/l test concentrations which showed measured concentrations of 72%, 138%, 79% and 78% respectively. Given that the 1.8 and 5.6 mg/l test concentrations were below the No Observed Effect Concentration, the measured concentrations for 10 and 32 mg/l were only slightly lower than the 80% acceptance limit and that the overall mean measured concentration at 48 hours was 91% of nominal it was considered that these values did not affect the outcome of the study.

As the test organisms were exposed to a mixture of degradents, and toxicity cannot be attributed to a single component, the results are based on nominal test concentrations only.

Range-finding test
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in the below table (Table 1).

No immobilisation was observed at the test concentrations of 0.10 and 1.0 mg/l. However, immobilisation was observed at 10 and 100 mg/l.

Based on this information test concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l were selected for the definitive test.

The test preparations were observed to be clear, colourless solutions throughout the duration of the test.

- Mortality: No mortalities recorded.

- EC50/LC50:

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in the below table.

Inspection of the immobilisation data at 24 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)
24 >100 -
48 88 64 - 160


The No Observed Effect Concentrations after 24 and 48 hours exposure were 56 and 10 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slope and standard error of the response curve at 48 hours was 2.5 (SE = 0.56).

- Other:
Reported statistics and error estimates:
None recorded.

Any other information on results incl. tables

Nominal
Concentration
(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

0.10

0

0

1.0

0

0

10

0

2

100

0

7

Table 1 - Cumulative Immobilisation Data in the Range-finding Test

Nominal
Concentration
(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

1.0

0

0

0

0

0

0

0

0

1.8

0

0

0

0

0

0

0

0

3.2

0

0

0

0

0

0

0

0

5.6

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

18

0

0

0

0

0

1

1

5

32

0

0

0

0

1

2

3

15

56

0

0

0

0

3

3

6

30

100

2

2

4

20

6

5

11

55

Table 2 Cumulative Immobilisation Data in the Definitive Test

R1 – R2 = Replicates 1 and 2

Nominal
Concentration
(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

3 Hours

24 Hours

48 Hours

R1

R2

Total

%

R1

R2

Total

%

R1

R2

Total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

0.32

0

0

0

0

0

0

0

0

0

0

0

0

0.56

0

0

0

0

0

0

0

0

1

2

3

15

1.0

0

0

0

0

6

5

11

55

9

10

19

95

1.8

0

0

0

0

10

10

20

100

10

10

20

100

3.2

2

1

3

15

10

10

20

100

10

10

20

100

Table 3 Cumulative Immobilisation Data in the Positive Control

R1 – R2 = Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 88 mg/l with 95% confidence limits of 64 - 160 mg/l. The No Observed Effect Concentration at 48 hours was 10 mg/l.
Executive summary:

The 48-hr-acute toxicity of U-donor to Daphnia Magna was studied under semi-static conditions.  Daphnids were exposed to control and test chemical at nominal concentrations for 48hr.  Immobilization and sublethal effects were observed at test termination.  The 48-hour EC50 was 88 mg/l.  The 48-hr NOEC based on Immobilization was 10 mg/l                       . 

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Results Synopsis Test Organism Age: 1stinstar Test Type: Semi-static 95% C.L.: 64 ‑ 160 mg/l NOEC:  10 mg/l                        EC50:  88 mg/l