Salts of 2-ethylhexyl phosphate esters with (Z)- octadec-9-enylamine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-Oct-2018 to 02-Nov-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance OECD 202 guideline without deviation. The substance was a UVCB of varying solubilities and therefore a WAF approach has been used in accordance with OECD Series on testing and assessment No. 23. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Year: 2008

Deviations:

no

Principles of method if other than guideline:

- Principle of test: Water accomodated fraction (WAF) was used in accordance with OECD guidance on aquatic toxicity testing of difficult substances and mixtures (Document no. 23, 2002).
- Short description of test conditions: Each concentration is prepared separately, whereby the test item is added to the medium at a specific loading rate. The material is left until fully dissolved (or as dissolved as possible i.e. a saturated solution of the varying componnets of the UVCB), then particulate matter is left to settle out of the water column. The remaining substance in the water column of the sample is transferred to the test vessels. This is done at each concentration. The concentration-response relationship is based on the initial loading rate of the vessel.
- Parameters analysed / observed: Pariculate was monitored in the WAFs via microscopic examination.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Provided by Sponsor, Confidential
- Storage conditions - room temperature in the dark
- Expiration date of the lot/batch: Confidential

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature in the dark
- Stability under test conditions:
No significant change in measured test item analyte concentrations over 48 h (within 80-120 % of nominal). However, stability of the entire UVCB is unknown.
- Solubility and stability of the test substance in the solvent/vehicle:
Not applicable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
None

Analytical monitoring:

yes

Remarks:

analytical monitoring used during both range finding and definitive tests, but a validated method was used during definitive test only

Details on sampling:

- Concentrations: 0.10, 0.18, 0.32, 0.56 and 1 mg/L
- Sampling method: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates (R1 to R4) at 48 hours for quantitative analysis. A set of duplicate samples was taken on each occasion and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A nominal amount of test item (10 mg) was added to the surface of 10 liters of culture medium to give rise to a 1.0 mg/L loading rate. This was stirred for 23 hours and allowed to settle for 1 hour. The aqueous phase was removed by mid-depth siphoning (~5 cm) with the first 75 - 100 mL discarded. This gave rise to a 1 mg/L (nominal) WAF stock. This was subsequently used to make successive dilutions (spacing factor ~1.8) to achieve 0.56, 0.32, 0.18 and 0.10 mg/L.
- Eluate: N/A
- Differential loading: N/A
- Controls: Elendt M7 (OECD 202, 2004)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Microscopic inspection of the WAF showed no micro-dispersions of test item to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: N/A in-house culture
- Justification for species other than prescribed by test guideline: N/A test guideline species used.
- Source: in-house (ENVIGO - Shardlow, UK)
- Age of parental stock (mean and range, SD): Not known, first instar neonates used suggesting 8-12 days old.
- Feeding during test: no
- Food type: N/A
- Amount: N/A
- Frequency: N/A

ACCLIMATION
- Acclimation period: not required, test and housing medium were the same.
- Acclimation conditions (same as test or not): N/A
- Type and amount of food: Desmodesmus subspicatus (algae) and GM 300 fish feed suspension.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): N/A

QUARANTINE (wild caught)
- Duration: N/A
- Health/mortality: N/A

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: 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

Remarks:

Elendt M7 syntheitc medium

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

OECD 202 test guideline recommendation

Post exposure observation period:

No.

Hardness:

No data, but sufficient for the test, standard medium used

Test temperature:

21-22 °C

pH:

7.7-8.1

Dissolved oxygen:

8.7-9.7

Salinity:

N/A

Conductivity:

No data

Nominal and measured concentrations:

1.00, 0.56, 0.32, 0.18, 0.10 mg/L nominal.
0 h measured concentrations: 0.155, 0.0887, 0.0481, 0.0203, 0.0174 mg/L, respectively.
48 h measured concentrations: 0.161, 0.0896, 0.0480, 0.0204, 0.0166 mg/L, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type (delete if not applicable): covered to reduce evaporation
- Material, size, headspace, fill volume: 150 mL glass beakers filled with 100 mL media with ~50 mL headspace.
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): static testing methodolgy
- 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): N.A
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 as prescribed by OECD 202 TG (2004)
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: not reported
- Conductivity: N/A
- Salinity: N/A
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 h light, 8 hours dark
- Light intensity: between 890 and 944 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortlaity. Sub-lethal effects also monitored were: colour, mobility and surface entrapment.

VEHICLE CONTROL PERFORMED: N/A

RANGE-FINDING STUDY
- Test concentrations: 1.0, 0.10 and 0.01 mg/L
- Results used to determine the conditions for the definitive study: yes in conjunction with an initial test which spanned a greater number of concnetrations. The initial test concentrations 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L. The results of the initial experiment showed significant effects occurred in the 0.10 mg/L loading rate WAF test group and as such the test was repeated.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

0.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

0.47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Refer to Table 1 and 2 in other information.
- Observations on body length and weight: N/A
- Other biological observations: Refer to Table 1 and 2 in other information.
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: Some reduced mobility and surface entrapment of the organisms refer to Table 1 and 2 in other information.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitation as monitored by microscopic inspection.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, 24 hour EC50 within the expected range of 0.6 mg/L and 2.1 mg/L.
- Relevant effect levels: -
- Limit test: No
- Dose-response test: Yes, 5 concentrations tested 0.32, 0.56, 1.0, 1.8 and 3.2
- ECx: 24 hour EC50 1.1 mg/L 48 hour EC50 0.80 mg/L
- Other: -

Reported statistics and error estimates:

For the reference substance test analysis of the immobilization data was carried out using Weibull analysis using linear maximum-likelihood regression at 24 and 48 hours.

In the definitive test the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The LOEL and the NOEL at 24 and 48 hours were calculated using the Step-down Cochran-Armitage test and Step-down Rao-Scott-Cochran- Armitage test respectively. All results were calculated using the ToxRat Professional computer software package (TOXRAT).

The 24 hour EC50 and confidence limits, for the positive control were: 1.1 mg/L (CL: 0.95 - 1.3 mg/L)
The 48 hour EC50 and confidence limits, for the positive control were: 0.80 mg/L (CL: 0.12 - 1.5 mg/L)

The 24 hour EL50 and confidence limits, for the test item were: 0.47 mg/L (CL: not determinable)
The 48 hour EL50 and confidence limits, for the test item were: 0.41 mg/L (CL: 0.35 - 0.48 mg/L)

Table 1 % Immobilised Daphnia and observations during the test at 24 hours

	24 hours
Nominal loading rate (mg/L)	Mortality	Observations
Control	0	No adverse observations
0.10	0	No adverse observations
0.18	5	4 P 16 N
0.32	5	1 P 18 N
0.56	70	6 P 2 R 1 N
1.0	100	-

N = Normal
, P = Pale, 
R = Reduced mobility, 
T = Trapped at surface

Table 2 % Immobilised Daphnia and observations during the test at 48 hours

	48 hours
Nominal loading rate (mg/L)	Mortality	Observations
Control	0	No adverse observations
0.10	0	No adverse observations
0.18	5	6 P, 1 T, 13 N
0.32	15	3 P, 14 N
0.56	85	2 P, 1 T
1.0	100	-

N = Normal
, P = Pale, 
R = Reduced mobility, 
T = Trapped at surface

Table 3 Water quality measurements

	0 hours			24 hours	48 hours
Nominal Loading Rate (mg/L)	pH	mg O2/L	Temp °C	Temp °C	pH	mg O2/L	Temp °C
Control	8.1	9.7	21	21	7.7	8.9	21
0.10	8.0	9.1	21	21	7.9	8.8	21
0.18	7.9	9.0	21	21	7.9	8.9	21
0.32	8.0	8.9	21	21	8.0	8.8	21
0.56	8.0	8.9	21	21	8.0	8.7	21
1.0	8.0	8.9	21	21	8.0	8.8	21

Validity criteria fulfilled:

yes

Conclusions:

During a Daphnia magna acute toxicity test (OECD 202, 2004), the 24 and 48 hour EL50 of the test substance were 0.47 mg/ L (CL: not determinable) and 0.41 mg/ L (CL: 0.35 - 0.48 mg/L).

Executive summary:

The study was conducted in accordance with the OECD guideline 202 the “Daphnia sp. acute immobilisation test” (2004).  As the test item was a UVCB with substances unknown or of varying solubilities, a WAF approach was used in accordance with OECD series on testing and assessment no. 23 (2000).  

A range finding study was used to determine definitive test concentrations and optimal stirring and settling time for the creation of the WAFs at each concentration.  An initial test was conducted, but due to excessive toxicity this was used only to further refine the concentration range for the definitive.

The definitive test concentrations were, 0 (control), 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L. A single WAF was prepared by stirring the test item for 23 hours at room temperature and then leaving any particulate to settle for 1 hour. The concntratons were then made by serial dilution. Microscopic inspection of the WAFs showed no micro-dispersions of test item to be present. Water quality parameters were measured at the start, end and at 24 hours in 1 replicate. Analytical confirmation of the test substance was performed at each concentration at 0 fresh and 48 hours old.  All water quality parameters and study validity criteria were met as per OECD guideline 202. These were: No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water) and the dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels. A positive control (potassium dichromate) is routinely run at the laboratory proving sensitivity of the test organism. The reference was deemed acceptable.

The 48 hour NOELR was 0.18 mg/L

The 48 hour LOELR was 0.32 mg/L

The 24 hour EL50 and confidence limits, for the test item were: 0.47 mg/L (CL: not determinable)

The 48 hour EL50 and confidence limits, for the test item were: 0.41 mg/L (CL: 0.35 - 0.48 mg/L)

Description of key information

48h EL50 0.41 mg/L (CL: 0.35 - 0.48 mg/L); Daphnia magna; OECD 202, 2004; Vryenhoef, H., 2018.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.41 mg/L

Additional information

The study was conducted in accordance with the OECD guideline 202 the “Daphnia sp. acute immobilisation test” (2004).  As the test item was a UVCB with substances unknown or of varying solubilities, a WAF approach was used in accordance with OECD series on testing and assessment no. 23 (2000).  

The definitive test concentrations were, 0 (control), 0.10, 0.18, 0.32, 0.56 and 1.0 mg/L. Microscopic inspection of the WAFs showed no micro-dispersions of test item to be present. Water quality parameters were measured at the start, end and at 24 hours in 1 replicate. Analytical confirmation of the test substance was performed at each concentration at 0 fresh and 48 hours old.  All water quality parameters and study validity criteria were met as per OECD guideline 202. These were: No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water) and the dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels. A positive control (potassium dichromate) is routinely run at the laboratory proving sensitivity of the test organism. The reference was deemed acceptable.

The 48 hour NOELR was 0.18 mg/L

The 48 hour LOELR was 0.32 mg/L

The 24 hour EL50 and confidence limits, for the test item were: 0.47 mg/L (CL: not determinable)

The 48 hour EL50 and confidence limits, for the test item were: 0.41 mg/L (CL: 0.35 - 0.48 mg/L)