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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 July 25, 2011 to September 21, 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
RA study
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details on the read across justification. The short term toxicity study in aquatic invertebrates with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
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)
Specific details on test material used for the study:
Sponsor's identification: Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered)
CAS Number: 68307-94-8
Identifier: TIS O2891
Description: Amber coloured slightly viscous liquid
Batch number: CI1E0447 solvent free
Date received: 13 June 2011
Expiry date: 29 May 2013
Storage conditions: room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.The concentration and stability of the test substance in the test preparations were verified by chemical analysis at 0 and 48 h

- Sampling method: Water samples were taken from the control and each loading rate WAF test group (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis. Samples at the No Observed Effect Loading Rate and above only were analysed.

- Sample storage conditions before analysis: Samples were stored at approximately -20°C prior to analysis. Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
TEST WATER: The reconstituted water used for both the range-finding and definitive tests.

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

Preparation 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. 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-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Validation of mixing period: Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances. A WAF of nominal loading rate of 100 mg/L was prepared, in duplicate, in reconstituted water. One loading rate was stirred for a period of 23 h and the other for a period of 95 h. After a 1 h standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis.

Range finding study: Due to the low aqueous solubility and complex nature of the test item, for the purposes of the range-finding test the test substance was prepared as a Water Accommodated Fraction (WAF). Based on the results from the validation of mixing period trial, a preparation period of 24 hours was used. The loading rates 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 loading rates of 10 and 100 mg/l. Amounts of test item (25 and 250 mg) were separately added to the surface of 2.5 litres of reconstituted water to give the 10 and 100 mg/L loading rates respectively. After the addition of the test substance, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro particles of test item to be present.In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21ºC to 22ºC with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. 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. Each 250 ml test and control vessel contained 250 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded. The control group was maintained under identical conditions but not exposed to the test substance. A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test substance under test conditions. All samples were stored at approximately -20°C prior to analysis.

Definitive test: Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

Experimental preparation: Amounts of test item (25, 45, 80, 140 and 250 mg) were each separately added to the surface of 2.5 litres of reconstituted water to give the 10, 18, 32, 56 and 100 mg/L loading rates respectively. After the addition of the test substance, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 h. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nesco film was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no micro particles of test substance to be present. The concentration and stability of the test substance in the test preparations were verified by chemical analysis at 0 and 48 hours.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures. Adult Daphnia were maintained in 150 ml glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20°C. The lighting cycle was controlled to give a 16 h light and 8 h darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3
Test temperature:
22°C to 23ºC
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl. pH was recorded at the start and termination of the test. pH ranged from 7.9-8.1 at 0 hours and 7.8-8.0 at 48 hours.There were no treatment related differences for pH.
Dissolved oxygen:
Dissolved oxygen concentrations were recorded at the start and termination of the test. The oxygen concentration in some of the 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-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.
Salinity:
Freshwater used
Nominal and measured concentrations:
In the range-finding test daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/L. Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.
Details on test conditions:
EXPOSURE CONDITIONS: As in the range-finding test 250 mL glass jars containing approximately 250 mL of test preparation were used. At the start of the test 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 22ºC to 23ºC with a photoperiod of 16 h light and 8 h 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 substance. The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 h 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.

Vortex depth measrement: The vortex depth was recorded at the start and end of the mixing period.

Evaluation of data: An estimate of the EL50 values at 24 and 48 h was given by inspection of the immobilisation data.

Validation critera: The results of the test are considered valid if the following performance criteria are met:-In the control, not more than 10% of the daphnids appear to be immobilised or stressed after 48 h.

-The dissolved oxygen concentration at the end of the test should be greater than or equal to 3 mg/L in the control and test vessels.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Immobilisation
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
56 other: mg/L loading rate WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Immobilisation
Details on results:
Validation of mixing period: Pre-study investigational work indicated that there was no significant increase in the amount of total organic carbon by extending the preparation period for longer than 24 h. Therefore, for the purpose of testing the test substance was prepared using a stirring period of 23 h followed by a 1-Hour settlement period.

Range finding test: Cumulative immobilisation data from the exposure of Daphnia magna to the test substance during the range-finding test are given in Table 1 (see any other information on results incl. tables section). No immobilisation was observed at 10 mg/L loading rate WAF. However, immobilisation was observed at 100 mg/L loading rate WAF. Based on this information loading rates of 10, 18, 32, 56 and 100 mg/L, using a stirring period of 23 h followed by a 1-Hour standing period, were selected for the definitive test. Chemical analysis of the 10 and 100 mg/L loading rate WAFs showed measured concentrations of 0.319 and 5.11 at 0 h and 0.277 and 2.97 mg/L at 48 h. Despite a decrease in measured concentration at 100 mg/L loading rate WAF at 48 h, it was considered that the test substance was stable under test conditions given the results of the 10 mg/L loading rate WAF and those from the definitive test.

Definitive test: Immobilisation data: Cumulative immobilisation data from the exposure of Daphnia magna to the test substance during the definitive test are given in Table 2 (see Any other information on results incl. tables section). Inspection of the immobilisation data based on the nominal loading rates gave the following results: Time

EL*50 (mg/L) 24(h) >100 48 (h) >100
***EL=Effective loading rate**An EL50 value could not be calculated as less than 50% immobilisation occured at this time point. The No Observed Effect Loading rate after 24 and 48 h exposure was 56 mg/L loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.A single immobilised daphnid was observed at 10 mg/L loading rate WAF Replicate R2 after 48 h exposure. This was considered to be due to natural causes rather than a toxic effect given that no other immobilisation was observed at this loading rate and no immobilisation was observed at the higher loading rates of 18, 32 and 56 mg/L.

Physico-chemical measurement: Temperature was maintained at 22ºC to 23ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH. Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. 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.The oxygen concentration in some of the 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-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.

Vortex depth measurement: The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion (see Table 3 (see in any other information on results section).

Observations on test substance solubility: Observations on the test media were carried out during the mixing and testing of the WAFs. At the start of the mixing period the 10, 18, 32, 56 and 100 mg/L loading rates were observed to be colourless solutions with globules of test substance floating at the surface and dispersed throughout. After 23 h stirring the 10, 18, 32, 56 and 100 mg/L loading rates were observed to be colourless solutions with particles of test substance floating at the surface and dispersed throughout. After a 1-Hour standing period the 10, 18, 32, 56 and 100 mg/L loading rates were observed to be colourless solutions with particles of test substance floating at the surface, settled at the bottom of the stirring vessel and dispersed throughout. Microscopic examination of the WAFs showed microscopic particles of test substance to be present and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed no microscopic particles of test substance to be present. During the test the 10, 18, 32 and 56 mg/L loading rates were observed to be clear, colourless solutions. At 0 h the 100 mg/L loading rate WAF was observed to be a clear, colourless solution however, at 24 h the preparation was observed to be a slightly cloudy homogenous dispersion and after 48 h, a cloudy dispersion with test substance particles at the bottom of the vessel.

Chemical analysis of loading rates: Analysis of the 56 and 100 mg/L loading rate WAFs at 0 h showed measured concentrations of 12.0 and 8.36 mg/L respectively. It was considered that the result for the 56 mg/L loading rate WAF was erroneous given the results from the 100 mg/L loading rate WAF. Analysis of the duplicate sample, stored at approximately -20oC prior to analysis, showed a measured concentration of 6.08 mg/L indicating the initial result was erroneous. Analysis of the 56 and 100 mg/L loading rate WAFs at 48 h showed measured concentrations of 7.22 and 8.09 mg/L respectively. The dissolved test substance may have been one or several components of the test substance. Given that toxicity cannot be attributed to a single component or mixture of components but to the test substance as a whole, the results were based on nominal loading rates only.

Validation criteria:
1) In the control, no daphnia immobilisation was observed over the 48-Hour test period. Thus the validity criterion was met.
2) The oxygen content was ≥ 7.9 mg/L in the control and test vessels at the end of the test. Thus the validity criterion was met.
Results with reference substance (positive control):
Positive Control: A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

Exposure conditions for the positive control were similar to those in the definitive test with the exception of the temperatures during testing which ranged from 21°C to 22°C.

Analysis of the immobilisation data by the maximum-likelihood probit method at 24 and 48 h based on the nominal test concentrations gave the following results:
24 h EC50: 1.5 mg/L (95% confidence limits 1.3 - 1.8 mg/L)
48 h EC50: 0.99 mg/L (95% confidence limits 0.85 - 1.1 mg/L)
The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/L.
The No Observed Effect Concentration is based upon zero immobilisation at this concentration.The results from the positive control with potassium dichromate were within the normal range for this reference item.

Table1: Cumulative Immobilisation Data in the Range-finding Test

Nominal Loading Rate

(mg/L)

Cumulative Immobilised Daphnia
(Initial Population: 10 Per Replicate)

24 Hours

48 Hours

Control

0

0

10

0

0

100

2

2


Table 2: Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate

(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

10

0

0

0

0

0

1*

1*

5*

18

0

0

0

0

0

0

0

0

32

0

0

0

0

0

0

0

0

56

0

0

0

0

0

0

0

0

100

0

1

1

5

2

3

5

25

 

R1 -R2 = Replicates 1 and 2 * A single immobilised daphnid was observed at 10 mg/L loading rate WAF Replicate R2 after 48 hours exposure. This was considered to be due to natural causes rather than a toxic effect given that no other immobilisation was observed at this loading rate and no immobilisation was observed at the higher loading rates of 18, 32 and 56 mg/L. 

 

Table 3: Vortex Depth Measurements at the Start and End of the Mixing Period

 

Nominal Loading Rate (mg/L)

Control

10

18

*

+

*

+

*

+

Height of Water Column (cm)

14.5

14.5

14.5

14.5

15.0

15.0

Depth of Vortex (cm)

~ 0.2

~ 0.2

~ 0.2

~ 0.2

~ 0.2

~ 0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Nominal Loading Rate (mg/L)

32

56

100

*

+

*

+

*

+

Height of Water Column (cm)

14.5

14.5

14.5

14.5

15.0

15.0

Depth of Vortex (cm)

~ 0.2

~ 0.2

~ 0.2

~ 0.2

~ 0.2

~ 0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

Dimple present

 

* = Start of mixing period

+ = End of mixing period

Physico-Chemical Measurements

Nominal
Loading Rate
(mg/L)

0 Hours

24 Hours

48 Hours

pH

mg O2/L

%ASV*

T°C

TºC

pH

mg O2/L

%ASV*

T°C

Control

R1

8.0

8.8

101

22

22

7.9

8.4

97

22

 

R2

8.0

8.8

101

22

22

8.0

8.4

97

22

10

R1

8.0

8.7

100

22

22

7.9

8.2

94

22

 

R2

8.1

8.7

100

22

22

7.9

8.1

93

22

18

R1

7.9

8.6

99

22

22

7.9

8.2

94

22

 

R2

8.0

8.5

98

22

22

8.0

8.2

94

22

32

R1

7.9

8.6

99

22

22

7.9

8.2

94

22

 

R2

7.9

8.6

99

22

22

7.9

8.1

93

22

56

R1

7.9

8.4

97

22

22

7.9

8.1

93

22

 

R2

7.9

8.4

97

22

22

7.9

8.2

94

22

100

R1

8.0

8.4

98

23

22

7.8

8.0

92

22

 

R2

7.9

8.4

98

23

22

7.8

7.9

91

22

 

*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R1- R2= Replicates 1 and 2



Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the nominal 48 h EL50 of the substance in Daphnia magna was determined to be greater than 100 mg/L loading rate.
Executive summary:

An acute toxicity study was conducted to evaluate acute toxicity potential of the read across substance, phosphoric acid, mono- and di-C6 -10 -alkyl esters, in Daphnia magna according to OECD Guideline 202 and EU Method C.2, in compliance with GLP. Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals each) were exposed to Water Accommodated Fractions (WAFs) of the test substance over a range of nominal loading rates of 0, 10, 18, 32, 56 and 100 mg/L for 48 h at a temperature of 22 to 23ºC under static test conditions. Observations were performed at 0, 24 and 48 h to determine the % immobilised daphnids in the control and test vessels. Under the study conditions, the nominal 48 h EL50 of the substance in Daphnia magna was determined to be greater than 100 mg/L loading rate (Goodband and Mullee, 2012).

Description of key information

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

An acute toxicity study was conducted to evaluate acute toxicity potential of the read across substance, phosphoric acid, mono- and di-C6 -10 -alkyl esters, in Daphnia magna according to OECD Guideline 202 and EU Method C.2, in compliance with GLP. Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals each) were exposed to Water Accommodated Fractions (WAFs) of the test substance over a range of nominal loading rates of 0, 10, 18, 32, 56 and 100 mg/L for 48 h at a temperature of 22 to 23ºC under static test conditions. Observations were performed at 0, 24 and 48 h to determine the % immobilised daphnids in the control and test vessels. Under the study conditions, the nominal 48 h EL50 of the substance in Daphnia magna was determined to be greater than 100 mg/L (Goodband and Mullee, 2012).