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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 February to 20 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control.
- Frequency: at t=0 h and t=48 h
- Volume: 1.0 mL in True View Vials
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 1.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
The batch of Tributyl(ethyl) phosphonium diethylphosphate tested was a clear colourless liquid with a purity of 96.3% and was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions started with the highest concentration of 100 mg/L applying approximately 15 minutes of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
Since it was expected that part of the test item adsorbed to glass surfaces during development of the analytical method (Test Facility Study No. 20175974), test vessels were pre-incubated with test solutions prior to the exposure phase in order to saturate the glass surface with test item, herewith minimizing the loss of test item from solution during the exposure period. Test solutions were added to the respective test vessels and were statically incubated under test conditions for an overnight period in the combined limit/range-finding test and first full test, and for approximately three days in the final test. The period of pre-incubation in the final test was chosen for practical reasons and not intended for scientific purposes.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Species : Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- Reason for selection: This system has been selected as an internationally accepted invertebrate species.
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old

BREEDING
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures : After 7 days of cultivation half of the medium twice a week.
- Temperature of medium : 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
- Medium :M7, as prescribed by Dr. Elendt-Schneider

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
The hardness of the test medium was 180 mg/L (expressed as CaCO3).
Test temperature:
The temperature continuously measured in a temperature control vessel remained constant at 19°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within ±1°C).
pH:
The pH was measured at the beginning and at the end of the test, for all concentrations and the control. The measured pH in the test solutions at test initiation and at test termination ranged between 7.8 and 8.1. These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units).
Dissolved oxygen:
The dissolved oxygen content was measured at the beginning and at the end of the test, for all concentrations and the control. The measured oxygen content of the test solutions at test initiation and at test termination ranged from 9.3 to 9.7 mg/L. These test conditions remained within the limits prescribed by the study plan (>3 mg/L at the end of the test).
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: 2.2, 4.6, 10, 22, 46 and 100 mg/L.
Measured concentrations: The measured concentrations were at the level of nominal throughout the test, i.e. varied between 94 – 108% relative to nominal (see also Table 1 below)
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL glass beakers containing 50 of test solution The glass beakers were pre-incubated with test solutions
- Type: open
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 replicates for all concentrations
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Feeding: the water fleas were not fed during the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-medium prepared in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands).
- Culture medium different from test medium: Water fleas were cultured in M7 medium. The test was performed in adjusted ISO-medium.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity: no data

EFFECT PARAMETERS MEASURED
The number of mobile and immobile water fleas and the number of water fleas showing sublethal effects was recorded 24 and 48 hours after test initiation. Immobile water fleas are those animals which are not able to swim within 15 seconds after gentle agitation of the test flask.

RANGE-FINDING STUDY
- Test concentrations: A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 100 mg/L in the limit test. In addition, ten daphnids per group (5 per replicate, duplicate) were exposed to 0.10, 1.0 and 10 mg/L in the combined range-finding test.
- Results used to determine the conditions for the definitive study: No immobility was observed in the control and up to nominally 1.0 mg/L throughout the test. At the end of the test, 90 and 100% immobility was observed at nominal concentrations of 10 and 100 mg/L, respectively. Therefore, the expected 48h-EC50 was between nominally 1.0 and 10 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
9.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95%-CI: 8.5-12 mg/L
Details on results:
- Mortality of control: No immobility was observed in the control and up to nominally 4.6 mg/L throughout the exposure period, while 55% immobility was observed at nominally 10 mg/L after 48 hours of exposure. In addition, all daphnids exposed to nominally 22 and 46 mg/L were immobilized at the end of the test.
- Other adverse effects control: no adverse effect observed in the control
- Abnormal responses: A first full test was performed based on the results of the preceding combined limit/range-finding test. Twenty daphnids per test group (four replicates, 5 daphnids per vessel) were exposed to an untreated control and to nominal concentrations of 1.0, 2.2, 4.6, 10 and 22 mg/L. Test procedures and conditions were similar as those applied in the final test. At the end of the test, no immobility was observed in the control and at the three lowest concentrations, while 30 and 25% immobility was found at concentrations of nominally 10 and 22 mg/L, respectively. Samples taken from nominally 10 and 22 mg/L were analysed on the cation of the test item. The measured concentrations were at the level of nominal throughout the test, i.e. varied between 93 and 105% relative to nominal, and thus, indicate proper preparation of test solutions. The recorded responses of the exposed daphnids were obviously less than in the combined limit/range-finding test, while analysed concentrations of the cation were at the level of nominal throughout both tests. Inconsistent immobility between tests might be caused by differences in concentrations of the anionic constituent, which was not analytically monitored. However, this was considered unlikely as the test item was completely soluble in test medium and biodegradation was only found after eight days of incubation (Test Facility Study No. 20175975). The exact reason for lower sensitivity of the daphnids towards the test item is therefore unknown. The responses recorded in this test were not sufficient for reliable determination of EC50-values and were not in agreement with what was expected based on the results of the combined limit/range-finding test. Therefore, a second full (and final) test was performed using a broader concentration series.
Results with reference substance (positive control):
The actual response in the reference test with potassium dichromate are within the ranges of expected responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River. The 24h EC50 was 0.84 mg/L (95% CI 0.73 - 0.97 mg/L); The 48h EC50 was 0.47 mg/L (95% CI 0.41 - 0.55 mg/L).
Reported statistics and error estimates:
Not applicable.

Table 1: Average Exposure Concentration Versus Nominal Concentration

Nominal concentration (mg/L)

Measured concentration

(mg/L)

t=0h

Measured concentration

(mg/L)

t=48h

0

0.000741

0.000711

2.2

2.16

2.24

4.6

4.97

4.71

10

10.6

10.8

22

20.6

21.2

46

48.4

44.6

100

108

103

1Estimated value, calculated by extrapolation of the calibration curve.

Maximum contribution to the samples was 0.023%. Origin of the test item response is unknown.

Table 2: Number of mobile water fleas during the test

Time (h)

Replicate

Tributyl(ethyl) phosphonium diethylphosphate

Nominal conc. (mg/L)

Control

2.2

4.6

10

22

46

100

0

A

5

5

5

5

5

5

5

B

5

5

5

5

5

5

5

C

5

5

5

5

5

5

5

D

5

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

20

 

24

A

0

0

0

0

2

2(1)

5

B

0

0

0

0

3

2(2)

5

C

0

0

0

0

2

1

5(4)

D

0

0

0

0

3(1)

1

5(3)

Total immobilised

0

0

0

0

10

6

20

Effect %

0

0

0

0

50

30

100

 

 

 

 

 

 

 

48

A

0

0

0

2

5

5

5

B

0

0

0

2

5

5

5

C

0

0

0

4

5

5

5

D

0

0

0

3

5

5

5

Total immobilised

0

0

0

11

20

20

20

Effect %

0

0

0

55

100

100

100

( ) – number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.



Validity criteria fulfilled:
yes
Conclusions:
The 48h-EC50 of Tributyl(ethyl) phosphonium diethylphosphate was 9.7 mg/L based on analytically confirmed nominal exposure concentrations.
Executive summary:

The acute toxicity of Tributyl(ethyl) phosphonium diethylphosphate to water fleas (Daphnia magna) was tested following the OECD testing Guideline 202 (2004) and according to the GLP Guidelines.

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to nominal concentrations of 2.2, 4.6, 10, 22, 46 and 100 mg/L.Since it was expected that part of the test item adsorbed to glass surfaces,test vessels were pre-incubated with test solutions prior to the exposure phase in order to saturate the glass surface with test item, herewith minimizing the loss of test item from solution during the exposure period.The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

The measured concentrations were at the level of nominal throughout the test, i.e. varied between 94 – 108% relative to nominal. Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations. No immobility was observed in the control and up to nominally 4.6 mg/L throughout the exposure period, while 55% immobility was observed at nominally 10 mg/L after 48 hours of exposure. In addition, all daphnids exposed to nominally 22,46 and 100 mg/L were immobilized at the end of the test. The study met the acceptability criteria prescribed by the study plan and was considered valid. In conclusion, the 48h-EC50 for Daphnia magna exposed to Tributyl(ethyl) phosphonium diethylphosphate was 9.7 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 8.5 and 12 mg/L).

Description of key information

The acute toxicity of Tributyl(ethyl) phosphonium diethylphosphate to Daphnia magna was investigated in a 48 hour test according to OECD Guideline No. 202 (2004) and according to the GLP Guidelines.

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to nominal concentrations of 2.2, 4.6, 10, 22, 46 and 100 mg/L

Under the conditions of this study, a 48h EC50 of 9.7 mg/L was obtained based on analytically confirmed nominal concentrations (95% confidence interval between 8.5 and 12 mg/L)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
9.7 mg/L

Additional information