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:
from 2012-08-01 to 2012-2012-06-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with international standard guidelines under GLP conditions. The study report was well documented with all mandatory information included.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2011-08-31

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report):TEPA (Triethyl Phosphono Acetate)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control 1.0, 3.2, 10, 32 and 100 mg/L test groups (replicates R1-R4 pooled) at 0 and 48h for quantitative analysis.All 0-hour samples were stored at approximately -20°C prior to analysis
Duplicate samples , and samples of the 1.8, 5.6, 18 and 56 mg/L test groups were taken and stored at approximately -20 °C for further analysis if necessary

Test solutions

Details on test solutions:
The test item was dissolved directly in water

An amount of test item (250 mg) was dissolved in reconstituted water and the volume adjusted to 2.5 litters to give 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 56, 32, 18, 10, 5.6, 3.2 1.8 and 1.0 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity

The concentration of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name:Daphnia magna
- Strain: No data
- Source: Derived from in-house laboratory cultures
- Age at study initiation (mean and range, SD): less than 24h old
- Method of breeding: In-house laboratory. Culture conditions ensure that reproduction was by parthenogenesis
- Feeding during test: No

ACCLIMATION
- Acclimation period: No
- Type and amount of food: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension
- Feeding frequency:no data
- Health during acclimation (any mortality observed): No data

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
250 mg/L of CaCO3
Test temperature:
21°C
pH:
7.9 to 8.1
Dissolved oxygen:
0h: 9.2 to 9.4 and 48h: 7.9 to 8.1
Salinity:
not applicable
Nominal and measured concentrations:
nominal concentrations: 0, 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L
Measured concentration: See results tables
Details on test conditions:
TEST SYSTEM
- Test vessel: glass jars
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL filled at 200mL
- Aeration: No
- 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):4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water:
Stock solution :
CaCl2 . 2H2O 1.76 g/L, MgSO4 . 7H2O 4.93 g/L, NaHCO3 : 2.59 g/L, KCl : 0.23 g/L
Preparation
An aliquot (25mL) of each solution a-d was added to each litre (Final volume) of deionized water with a conductivity of <5µS/cm. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-satured value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3
- Conductivity: 5µS/cm

OTHER TEST CONDITIONS
- Adjustment of pH: yes if necessary
- Photoperiod: 16hours light and 8hours darkness cycle with 20 minute dawn and dusk transition period
- Light intensity: 903 to 990 Lux over the test period

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :immobilization measured every day

TEST CONCENTRATIONS
- Test concentrations: 0, 1.0, 1.58, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L
- Justification for using less concentrations than requested by guideline:
- Range finding study; Yes
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No immobilization at 0.1 and 1.0 mg/L. However 10 and 100 mg/L, immobilization was observed
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate (Study number: 41104946)

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 3.6-4.3 mg/L
Details on results:
Cumulative immobilization data from the exposure of Dapnia Magna to the test item are given in table 2.

The EC50 after 24h and 48h exposure were 7.3 (6.9-7.7) and 3.9 (3.6-4.3) respectively
The NOEC after 24h and 48h exposure were 3. 2mg/L and 1.8 mg/L respectively. Due to unsuitable nature of the data, it was not possible to calculate the slope and error of response curve at 24 and 48 hours.

All control and test preparations were observed to be clear colourness solutions throughout the duration of the test.

Temperature was maintained at 20°C to 21°C throughout the test, whilst the light intensity was observed to range from 903 to 990 lux over test period. There were no treatment related differences for oxygen concentration or pH.

The oxygen concentration in some of test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturing the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effect were observed on the control group.

Analysis of the test preparation at 0 and 48 hours showed measured test concentrations to be near nominal with exception of the 32 mg/L test sample at 48 hours which showed a measured test concentration of 76% of nominal was obtained. As the overall measured concentrations were within 80 to 120 % of nominal, it was considered appropriate to calculate the results based on the nominal test concentrations only.
Results with reference substance (positive control):
The study was conducted between29 November 2011 and 01 December 2011.
concentration: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed Spearman-Karber method at 48 hours based on the nominal test concentration gave the following results:
The EC50 after 24h and 48h exposure were 1.3 (1.0-1.8) and 1.4 (1.0-1.3) respectively
The NOEC after 24h and 48h exposure were 1.0 mg/L and 0.56 mg/L respectively.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

Table 2 : Cumulative immobilisation data

 

Nominal concentration

(mg/L)

Cumulative immobilised Daphnia (initial population : 5 per replicate)

24 hours

48 hours

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Total

%

Control

0

0

0

0

0

0

0

0

0

0

0

0

1.0

0

0

0

0

0

0

0

0

0

0

0

0

1.8

0

0

0

0

0

0

0

0

0

0

0

0

3.2

0

0

0

0

0

0

1

1

1

0

3

15

5.6

1

0

0

0

5

5

5

5

5

5

20

100

10

5

5

5

5

20

100

5

5

5

5

20

100

18

5

5

5

5

20

100

5

5

5

5

20

100

32

5

5

5

5

20

100

5

5

5

5

20

100

56

5

5

5

5

20

100

5

5

5

5

20

100

100

5

5

5

5

20

100

5

5

5

5

20

100

R1 – R4 : Replicates 1 to 4

 

Concentrations of test item in aqueous test media samples determined using HPLC-MS Analysis

 

Sample

Nominal concentration

(mg/L)

Concentration found

(mg/L)

Expressed as a percentage of the nominal concentration (%)

0 hours

Control

<LOQ

-

1.0

0.898

90

3.2

2.79

87

10

8.66

87

32

23.6

82

100

87.8

88

48 hours

Control

<LOQ

-

1.0

0.834

83

3.2

2.70

84

10

8.14

81

32

24.5

76

100

82.0

82

 

Physico-chemical measurements

 

Nominal concentration

(mg/L)

0 hours

24 hours

48 hours

pH

Mg O2/L

% ASV *

T (°C)

T

 (°C)

pH

Mg O2/L

% ASV *

T

(°C)

Control

R1

8.0

9.3

102

20

21

8.1

9.1

102

21

R2

8.0

9.3

102

20

21

8.1

9.1

102

21

R3

8.0

9.3

102

20

21

8.1

9.0

101

21

R4

8.0

9.3

102

20

21

8.1

9.0

101

21

1.0

R1

8.0

9.3

102

20

21

8.1

9.0

101

21

R2

8.0

9.3

102

20

21

8.1

8.9

100

21

R3

8.0

9.3

102

20

21

8.1

9.0

101

21

R4

8.0

9.3

102

20

21

8.1

8.9

100

21

1.8

R1

8.0

9.3

102

20

21

8.1

8.9

100

21

R2

8.0

9.3

102

20

21

8.1

8.9

100

21

R3

8.0

9.3

102

20

21

8.1

8.9

100

21

R4

8.0

9.3

102

20

21

8.1

8.9

100

21

3.2

R1

8.0

9.3

102

20

21

8.0

8.9

100

21

R2

8.0

9.3

102

20

21

8.1

8.9

100

21

R3

8.0

9.2

101

20

21

8.1

8.9

100

21

R4

8.0

9.3

102

20

21

8.1

8.9

100

21

5.6

R1

8.0

9.3

102

20

21

8.0

8.9

100

21

R2

8.0

9.3

102

20

21

8.0

8.9

100

21

R3

8.0

9.3

102

20

21

8.0

8.9

100

21

R4

8.0

9.3

102

20

21

8.0

8.9

100

21

10

R1

8.0

9.3

102

20

21

8.0

8.9

100

21

R2

8.0

9.3

102

20

21

8.0

8.9

100

21

R3

8.0

9.3

102

20

21

8.0

8.8

99

21

R4

8.0

9.3

102

20

21

8.0

8.9

100

21

18

R1

8.0

9.4

103

20

21

8.0

8.8

99

21

R2

8.0

9.3

102

20

21

8.0

8.8

99

21

R3

8.0

9.3

102

20

21

8.0

8.8

99

21

R4

8.0

9.4

103

20

21

8.0

8.8

99

21

32

R1

8.0

9.3

102

20

21

8.0

8.8

99

21

R2

8.0

9.3

102

20

21

8.0

8.8

99

21

R3

8.0

9.3

102

20

21

8.0

8.8

99

21

R4

8.0

9.4

103

20

21

8.0

8.8

99

21

56

R1

8.0

9.3

102

20

21

8.0

8.8

99

21

R2

8.0

9.3

102

20

21

8.0

8.8

99

21

R3

8.0

9.3

102

20

21

8.0

8.8

99

21

R4

8.0

9.3

102

20

21

8.0

8.8

99

21

100

R1

8.0

9.3

102

20

21

8.0

8.8

99

21

R2

8.0

9.3

102

20

21

7.9

8.7

98

21

R3

8.0

9.2

101

20

21

8.0

8.7

98

21

R4

8.0

9.3

102

20

21

7.9

8.7

98

21

* ASV = Dissolved oxygen concentration expressed as a percentage of air saturation value

R1 – R4 : Replicates 1 to 4

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia Magna has been investigated and gave a 48-hour EC50 value of 3.9mg/L. 95% confidence limits 3.6-4.3 mg/L. The NOEC at 48 hours was 1.8 mg/L
Executive summary:

The 48–hr-acute toxicity of TEPA to Daphnia magna was studied under static conditions.  Daphnids were exposed to control and test chemical at measured concentration of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/L for 48 hr.  Mortality were observed daily. 

The 48– hour EC50was > 3.9 mg/L. (3.6 -4.3 mg/L)

 

It satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

 

Results Synopsis

 Test Organism :Daphnia magna

 NOEL:  1.8 mg.//L   

EC5O: > 3.9 mg/L