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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-20 - 2018-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines, OCSPP 850.1010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch/Lot Number: 7703200
Description: clear, pale yellow liquid
Purity: considered as 100%
Manufacture date: 04 February 2016
Expiry date: 28 February 2019
Storage condition: Controlled room temperature (15-25°C, below 70 RH%)
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were performed from the control and at the applied test concentration level at the beginning and at the end of the experiment.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because the test item is very poorly soluble in water, a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
A saturated test item solution (nominal loading rate of 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (ISO Medium) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C.
The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100 mg/L nominal loading rate WAF.
As a Limit test was carried out, further dilution of stock solution was not performed.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary; The Daphnia are bred in Ecotoxicological Laboratory of Citoxlab Hungary Ltd. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
- Age of the animals: <24 h old at the beginning of the test

ACCLIMATION
- Acclimation period: There was no acclimatization because the water used was similar to the culture water.

Number of animals: There were 20 animals in test group and control group respectively, divided into 4 replicates (5 animals / replicate)

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
248 mg/L (as CaCO3)
Test temperature:
20.5 – 21.0°C
pH:
7.00 – 7.41
Dissolved oxygen:
6.9 – 8.5 mg/L
Salinity:
Not available
Conductivity:
Not available
Nominal and measured concentrations:
Nominal loading rate = 100 mg/L
Geometric mean measured concentration = 5.62 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Material, size, headspace, fill volume: 50 mL; 40 mL test solution
- Aeration:
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 8 mL test solution/animal

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.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16-hour light/8-hour dark cycle
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilisation - 24 & 48 hours.

VEHICLE CONTROL PERFORMED: No.

RANGE-FINDING STUDY
- Test concentrations: 0.1, 1, 10, 100 % saturated solution
- Results used to determine the conditions for the definitive study: 0.1% - 1/10 immobilised; 1% - 0/10 immoblised; 10% - 0/10 immobilised; 100% - 1/10 immobilised.
Because toxic response was not observed during the preliminary concentration rangefinding test, a Limit Test was carried out using only one test concentration at the solubility level of the test item (nominal loading rate of 100 mg/L) and one control group in a static system.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 5.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
5.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: none observed
- Mortality of control: no mortality/immobilisation observed
- Other adverse effects control: none reported
- Abnormal responses: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
The date of the last study (Study Code: 17/058-023DA) with reference item Potassium dichromate (batch no.: A0345704) is: 08 - 09 March 2017.
The 24h EC50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.68 mg/L)
Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects.
The EC50, NOEC, LOEC and EC100 values were determined directly from the raw data.

No immobilised animals were recorded in the control or test item groups at 24 or 48 hours.

In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Measured test concentrations:

Control: Start - none detected; End - none detected

100% saturated solution: Start - 4.83 mg/L; End - 6.55 mg/L

Geometric mean measured concentration = 5.62 mg/L

Validity criteria fulfilled:
yes
Remarks:
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.
Conclusions:
The 24h and 48h EC50 value: > 5.62 mg/L (measured)
The 48h No-Observed Effect Concentration (NOEC): 5.62 mg/L (measured)
Executive summary:

Acute toxicity of Phosphoric acid, C8-12 (even numbered) alkyl esters on Daphnia magna was assessed in an Acute immobilisation test, over an exposure period of 48 hours in a static test system (OECD TG 202). Because no toxic response was observed during the preliminary range-finding test, a Limit Test was carried out using only one test concentration at the solubility level of the test item (100 mg/L nominal loading rate WAF) and one control group in the definitive test.

The test concentration was analytically determined at the start and at the end of the experiment. Measured concentration was 4.83 mg/L at the start and 6.55 mg/L at the end of the experiment, thus the corresponding measured geometric mean test item concentration was 5.62 mg/L. The biological results are based on the measured geometric mean test item concentration.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentration and for the control.

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 Phosphoric acid, C8-12 (even numbered) alkyl esters were the followings:

The 24h and 48h EC50 value: > 5.62 mg/L (measured)

The 48h EC100 value: > 5.62 mg/L (measured)

The 48h No-Observed Effect Concentration (NOEC): 5.62 mg/L (measured)

The 48h Lowest Observed Effect Concentration (LOEC): > 5.62 mg/L (measured)

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was conducted between 7 June 2011 and 25 June 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Principles of method if other than guideline:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a 1-Hour settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.Validation of mixing period:Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF. A WAF of nominal loading rate of 100 mg/l was prepared, in duplicate, in deionised reverse osmosis purified water. One loading rate was stirred for a period of 23 hours and the other for a period of 71 hours. After a 1-Hour standing period the mixtures were then removed by siphon and the concentration of the test item in the 100 mg/l loading rate WAF was verified by chemical analysis (see details on analyitcal methods section).
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Single nominal loading rate of 100 mg/l.The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see details on analytical methods section).- Sampling method: Water samples were taken from the control (replicates R1 – R4 pooled) and the 100 mg/l loading rate WAF test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis. The method of analysis, recovery and test preparation analyses are described in details on analytical methods section.- Sample storage conditions before analysis:Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: Due to the low aqueous solubility and complex nature of the test item for the purposes of the definitive test the test item was prepared as a Water Accommodated Fraction (WAF).An amount of test item (250 mg) was added to the surface of 2.5 litres of reconstituted water to give the 100 mg/l loading rate. After the addition of the test item, 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 mixture allowed to stand for 1 hour. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.- Eluate: Not applicable- Controls: 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.- Evidence of undissolved material : Observations on the test media were carried out during the mixing and testing of the WAF.At the start of the mixing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with oily globules of test item dispersed throughout. At the end of the mixing period and following the 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with an oily layer of test item on the surface. Microscopic inspection of the WAF showed no microscopic particles of test item to be present. At the start and throughout the duration of the test all control and test preparations were observed to be clear colourless solutions.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 in any other information on results including tables section).
Test organisms (species):
Daphnia magna
Details on test organisms:
Test SpeciesThe 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 hours light and 8 hours 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:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.
Test temperature:
Temperature was maintained at 21 to 22ºC throughout the test.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.Water temperature was recorded daily throughout the test. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HClThe pH levels were measured using a Hach HQ30d Flexi Handheld meter.There was no treatment related differences for pH.The results of the physico-chemical measurements are given in any other information on results including tables section.
Dissolved oxygen:
The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter.The results of the physico-chemical measurements are given in the any other information on results including tables section.
Salinity:
Freshwater used.
Nominal and measured concentrations:
Nominal: Single loading rate of 100 mg/l.Measured: Analysis of the test preparations at 0 hours (see details on analytical methods section) showed measured concentrations of of 96.8 mg/l and 97.2 mg/l in replicates R1 and R2 of the 100 mg/l loading rate WAF respectively. Measured concentrations of 96.4 mg/l and 98.2 mg/l were observed in replicates R1 and R2 respectively of the 100 mg/l loading rate WAF at 48 hours.Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.
Details on test conditions:
TEST SYSTEMAs in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21 to 22ºC 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 item.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. - Aeration: None.- Source/preparation of dilution water:Reconstituted Water – Elendt M7 MediumSolution Concentration of stock solution (mg/l)(I)H3BO357190(II)MnCl2.4H2O7210(III)LiCl6120(IV)RbCl1420(V)SrCl2.6H2O3040(VI)NaBr320(VII)Na2MoO4.2H2O1260(VIII)CuCl2.2H2O335(IX)ZnCl2260(X)CoCl2.6H2O200(XI)Kl65(XII)Na2SeO343.8(XIII)NH4VO311.5(XIV)Na2EDTA.2H2O5000 FeSO4.7H2O1991An aliquot (dependant on the volume of medium required) of each stock solution was added to a final volume of deionised reverse osmosis water to give stock solution A and stored at approximately 21ºC.Macro Nutrient Stock SolutionsSolution Concentration of stock solution (g/l)(I)CaCl2.2H2O293.80(II)NaHCO364.80(III)MgSO4.7H2O246.60(IV)Na2SiO3.9H2O50.00(V)KCl58.00(VI)NaNO32.74(VII)K2HPO41.84(VIII)KH2PO41.43Vitamin NutrientsSolution Concentration of stock solution (mg/l)(IX)Thiamine hydrochloride750Cyanocobalamine (vitaminB12) 100D(+) biotin (vitamin H)75The final medium was prepared by adding an aliquot of stock solution A along with aliquots of each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the required amount (final volume) of deionised reverse osmosis water. The pH of the prepared media was 8.0 ± 0.2 and stored at approximately 21ºC.Reconstituted Wateri)Stock Solutionsa)CaCl2.2H2O11.76 g/lb)MgSO4.7H2O4.93 g/lc)NaHCO32.59 g/ld)KCl0.23 g/lii)PreparationAn 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.- Culture medium different from test medium:Reconstituted water was used for both the range-finding and definitive tests. The reconstituted water is defined in above.EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 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 CONCENTRATIONS- Range finding study: Yes; The loading rate to be used in the definitive test was determined by a preliminary range-finding test. - Test concentrations: In the range-finding teest Daphnia magna were exposed to a series of nominal loading rates of 10 to 100 mg/l.. - Results used to determine the conditions for the definitive study: Based on the results of the range-finding test a 'limit test' was conducted at a single loading rate of 100 mg/l to confirm that no immobilisation or adverse reactions to exposure were observed.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 other: mg/l LR WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL not stated
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 other: mg/l LR WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% CL not stated
Details on results:
- 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: Observations on the test media were carried out during the mixing and testing of the WAF.At the start of the mixing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with oily globules of test item dispersed throughout. At the end of the mixing period and following the 1-Hour standing period the 100 mg/l loading rate WAF was observed to have formed a clear colourless water column with an oily layer of test item on the surface. Microscopic inspection of the WAF showed no microscopic particles of test item to be present. At the start and throughout the duration of the test all control and test preparations were observed to be clear colourless solutions.
Results with reference substance (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. Analysis of the immobilisation data by the maximum-likelihood probit method (Finney 1971 ) at 24 and 48 hours based on the nominal test concentrations gave the followingresults:Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)24 1.5 1.3 - 1.848 0.99 0.85 - 1.1The 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.

Range-finding Test

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table1 (below).

No immobilisation was observed at 10 and 100 mg/l loading rate WAF. 

Based on this information, a single loading rate of four replicates, of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.

Chemical analysis of the test preparations at 0 and 48 hours (see details on analytical methods section) gave measured concentrations of 97.7 mg/l and 101 mg/l at 0 and 48 hours respectively. Only samples at the No Observed Effect Loading Rate (NOEL) and above were analysed.

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

0

0

Definitive Test Immobilisation data

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours.

 

Table 2              Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate

(mg/l)

Cumulative Immobilised Daphnia
(Initial Population: 5 Per Replicate)

24 Hours

48 Hours

No. Per

Replicate

Total

%

No. Per

Replicate

Total

%

Control

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0

100

R1

0

0

0

0

0

0

 

R2

0

0

 

R3

0

0

 

R4

0

0

 

R1– R4= Replicates 1 to 4

There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours. Inspection of the immobilisation data gave the following results:

24 hr EL50 (mg/l): >100

48 hr EL50 (mg/l): >100

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

Vortex Depth Measurements

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).

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

 

Nominal Loading Rate (mg/l)

Control

100

*

+

*

+

Height of Water Column (cm)

14.8

14.8

14.7

14.7

Depth of Vortex (cm)

~0.2

~0.2

~0.2

~0.2

Observation of Vortex

Dimple present

Dimple present

Dimple present

Dimple present

 


*= Start of mixing period

+= End of mixing period

Validation of Mixing Period

Pre-study investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher measured levels of test item, in the WAF. A WAF of a nominal loading rate of 100 mg/l was prepared in duplicate in deionised reverse osmosis purified water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 71 hours. After a 1-Hour standing period the mixtures were then removed by siphon and samples taken for chemical analysis. 

The results are summarised as follows:

Test Item Loading Rate

(mg/l)

Measured Concentration (mg/l)

Time (hours)

24

72

Control

<LOQ

<LOQ

100

59.3

82.9

Although the results from this pre-study investigational work showed that the measured concentration increased from 24 hours to 72 hours, chemical analysis of the 100 mg/l Loading rate WAF test samples from the range-finding test showed that 24 hours was a sufficientpreparation period to ensure the maximum amount of the dissolved water soluble fraction of the test item. The preparation of the WAF was maintained at 24 hours.


 LOQ = Limit of Quantitation (assessed down to 0.03 mg/l)

Physico-chemical measurements

The results of the physico-chemical measurements are given below.

Temperature was maintained at 21 to 22°C 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.

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

7.9

8.8

99

21

22

8.2

8.7

100

22

 

R2

7.9

8.8

99

21

22

8.2

8.7

100

22

 

R3

7.9

8.7

98

21

22

8.1

8.7

100

22

 

R4

7.9

8.7

98

21

22

8.1

8.6

99

22

100

 

R1

7.6

8.7

98

21

22

8.0

8.4

97

22

R2

7.5

8.6

97

21

22

7.9

8.4

97

22

R3

7.5

8.6

97

21

22

7.8

8.4

97

22

R4

7.5

8.6

97

21

22

7.7

8.4

97

22

 


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

R1– R4= Replicates 1 to 4

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 EL*50 value of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.* EL = Effective Loading rate
Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 21 to 22ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results.

The 48-Hour EL*50 for the test item to Daphnia magnab ased on nominal loading rates was greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Information supplied by the Sponsor, indicated that the test item contained approximately 42% 2-ethylhexyl dihydrogen phosphate (MW = 210 g/mol) and 49% bis(2-ethylhexyl) hydrogen phosphate (MW = 322 g/mol)). Under the acidic conditions of the analysis, both compounds could be observed, but the lower acidity and greater response of the bis(2-ethylhexyl) hydrogen phosphate meant that it appeared as the main peak in the chromatogram.

Analysis of the test preparations at 0 hours showed measured concentrations of of 96.8 mg/l and 97.2 mg/l in replicates R1 and R2 of the 100 mg/l loading rate WAF respectively. Measured concentrations of 96.4 mg/l and 98.2 mg/l were observed in replicates R1 and R2 respectively of the 100 mg/l loading rate WAF at 48 hours.

Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.


*EL = Effective Loading rate

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis proposed is that the organism is not exposed to common compounds but rather, because of structural similarity, that different compounds have similar ecotoxicological and fate properties. In this case the ECHA Read-Across Assessment Framework (RAAF) Scenario 2 is used.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source-A: Phosphoric acid, C8-12 (even-numbered) alkyl esters [EC not yet assigned; CAS not assigned]
Source-B: Phosphoric acid, 2-ethylhexyl ester [EC 235-741-0; CAS 12645-31-7]
Target: Phosphoric Acid, Esters with Alcohols, C11-14 iso, C13-rich [EC not yet assigned; CAS not assigned]
3. ANALOGUE APPROACH JUSTIFICATION
Available data for the Source-A substance suggest that algae are the most sensitive species. This is confirmed by data available for fish, daphnia and algae from studies performed on the Source-B substance. In the available studies the effect levels in daphnia and algae are comparable between the substances. In this case, it is acceptable to read-across for the short-term toxicity to aquatic invertebrates endpoint from the Source-A and Source-B substances to the Target.
4. DATA MATRIX
Short-term toxicity to fish:
Source-A: No data
Source-B: 96-h LL50 value of greater than 100 mg/L loading rate WAF
Target: No data
Short-term toxicity to aquatic invertebrates:
Source-A : Daphnia magna; OECD TG 202.
48-h EC50 > 5.62 mg/L (measured) (100 mg/L nominal loading rate WAF)
48-h NOEC = 5.62 mg/L (measured) (100 mg/L nominal loading rate WAF)
Source-B: Daphnia magna; OECD TG 202.
48-h EL50 >100 mg/L loading rate WAF
48-h NOELR = 100 mg/L
Target: No data
Toxicity to aquatic algae:
Source-A: Pseudokirchneriella subcapitata; OECD TG 201
Results based on initial nominal loading rates (WAFs):
72 -h ErL50 > 100 mg/L; 72 -h EγL50 = 37.8 mg/L; 72 -h EbL50 = 36.51 mg/L
72 -h NOErLR = 12.5 mg/L; 72 -h NOEγLR = 6.25 mg/L; 72 -h NOEbLR = 6.25 mg/L
Results based on initial measured concentrations:
72 -h ErC50 > 28.92 mg/L; 72 -h EγC50 = 15.70 mg/L; 72 -h EbC50 = 15.29 mg/L
72 -h NOErC = 6.53 mg/L; 72 -h NOEγC = 3.71 mg/L; 72 -h NOEbC = 3.71 mg/L
Source-B:
72-h ErL50 = 49 mg/L loading rate WAF
72-h NOErLR = 25 mg/L
Target: No data
Reason / purpose:
read-across source
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 5.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
5.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility

Description of key information

The 24h and 48h EC50 value: > 5.62 mg/L (measured)

The 48h No-Observed Effect Concentration (NOEC): 5.62 mg/L (measured)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
5.62 mg/L

Additional information

No data are available for Phosphoric Acid, Esters with Alcohols, C11-14 iso, C13-rich. Acute toxicity of Phosphoric acid, C8-12 (even numbered) linear alkyl esters on Daphnia magna was assessed in an Acute immobilisation test, over an exposure period of 48 hours in a static test system (OECD TG 202). Because no toxic response was observed during the preliminary range-finding test, a Limit Test was carried out using only one test concentration at the solubility level of the test item (100 mg/L nominal loading rate WAF) and one control group in the definitive test.

The test concentration was analytically determined at the start and at the end of the experiment. Measured concentration was 4.83 mg/L at the start and 6.55 mg/L at the end of the experiment, thus the corresponding measured geometric mean test item concentration was 5.62 mg/L. The biological results are based on the measured geometric mean test item concentration.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentration and for the control.

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 Phosphoric acid, C8-12 (even numbered) alkyl esters were the followings:

The 24h and 48h EC50 value: > 5.62 mg/L (measured)

The 48h EC100 value: > 5.62 mg/L (measured)

The 48h No-Observed Effect Concentration (NOEC): 5.62 mg/L (measured)

The 48h Lowest Observed Effect Concentration (LOEC): > 5.62 mg/L (measured)

A study is also available for the 2 -ethylhexyl analogue. This study was performed according to OECD TG 202 and EU Method C.2.Following a preliminary range-finding test twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accomodated Fraction (WAF) of the test item at a single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 to 22 deg C under static test conditions. Immobilisation and any adverse reactions were recorded after 24 and 48 hours.

The 48-Hour EL*50 for the test item to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Information supplied by the Sponsor, indicated that the test item contained approximately 42% 2-ethylhexyl dihydrogen phosphate (MW = 210 g/mol) and 49% bis(2-ethylhexyl) hydrogen phosphate (MW = 322 g/mol)). Under the acidic conditions of the analysis, both compounds could be observed, but the lower acidity and greater response of the bis(2-ethylhexyl) hydrogen phosphate meant that it appeared as the main peak in the chromatogram.

Analysis of the test preparations at 0 hours showed measured concentrations of of 96.8 mg/L and 97.2 mg/L in replicates R1 and R2 of the 100 mg/L loading rate WAF respectively. Measured concentrations of 96.4 mg/L and 98.2 mg/L were observed in replicates R1 and R2 respectively of the 100 mg/L loading rate WAF at 48 hours.

Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole the results were based on nominal loading rates only.


*EL = Effective Loading rate