Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

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:

Between 27 October 2011 and 25 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP; appropriate WAF in agreement with guideline and generally accepted recommendations

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

Remarks:

WAF following OECD STA 23 (2000) and ECETOC Monograph 26 (1996) guidelines and considering literature recommendations (Singer et al 2002)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Remarks:

see EU C.2 entry

Qualifier:

according to guideline

Guideline:

other: OECD STA 23 (2000); Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Environmental Health and Safety Publications, Series on Testing and Assessment No. 23. Document Reference ENV/JM/MONO(2000)6.

Deviations:

not applicable

Remarks:

Appropriate elements considered

Qualifier:

according to guideline

Guideline:

other: ECETOC European Centre for Ecotoxicology and Toxicology of Chemicals (1996). Aquatic Toxicity Testing of Sparingly Soluble, Volatile and Unstable Substances. Monograph no. 26

Deviations:

not applicable

Remarks:

Appropriate elements considered

Qualifier:

according to guideline

Guideline:

other: Singer MM, Aurand D, Bragin GE, Clark JR, Coelho ML, Sowby ML, Tjeerdema RS (2000). Standardization of the Preparation and Quantitation of Water-Accommodated Fractions of Petroleum for Toxicity Testing. Marine Pollution Bulletin 40(11):1007-16.

Deviations:

not applicable

Remarks:

Appropriate elements considered

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 Monograph no. 26, OECD STA 23 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. Pre-study work showed that a preparation period of 96 h was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing and following a 1-h standing period, the test item phase was 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).

GLP compliance:

yes

Remarks:

Department of Health of Government of the U.K., inspection 19-21 July 2011

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (replicates R1 – R4 pooled) and 100 mg/L loading rate (replicates R1 – R2 and R3 – R4 pooled)
- Sampling method: Duplicate samples were taken at 0 and 48 h
- Sample storage conditions before analysis: At approximately -20 °C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test item (250 mg) was added to the surface of 2.5 L 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 95 h and the mixture allowed standing for 1 h. 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.
- Controls: Microscopic observations for dispersed test item were performed before and after filtration. The stirring period was monitored by measuring the height of the water column and the vortex depth at the start and end of the mixing period and by observation of vortex for dimple at the water surface on each occasion.
- Evidence of undissolved material: At the start of the mixing period the 100 mg/L loading rate was observed to be a clear, colourless water column with white globules floating on the surface. After 95 h stirring and a 1-h standing period the 100 mg/L loading rate was observed to remain a clear, colourless water column with white globules floating on the surface. After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear, colourless solution. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: In-house laboratory culture
- Age at study initiation: 1st instar daphnids less than 24 h old
- Method of breeding: Adult daphnids 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 min 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 h old. These young were removed from the cultures and used for testing.
- Feeding during test: No

ACCLIMATION
- Acclimation period: Not required as the breeding and test conditions were comparable

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The reconstituted water used to prepare the test media had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

21 to 22 °C in control and sole treatment replicates, maintained in a temperature controlled room
The values were recorded daily throughout the test using a Hanna Instruments HI 93510 digital thermometer.

pH:

7.7 to 8.1 (control) and 7.8 to 8.1 (sole treatment replicates)
The values were recorded daily throughout the test in the freshly prepared test preparations and the used or 24-h old test preparations using a Hach HQ30d Flexi Handheld meter.

Dissolved oxygen:

8.4 to 8.7 mg/L in control and sole treatment replicates
94 to 100 (control) and 94 to 99 (sole treatment replicates) % of Air Saturation Value
The values were recorded at the start and termination of the test using a Hach HQ30d Flexi Handheld meter.

Salinity:

Standard test media, therefore no salinity determination

Nominal and measured concentrations:

Nominal: One single loading rate of 100 mg/L and a control (0 mg/L)
Measured: In all control and treatment samples the measured concentration was < LOQ, which was determined to be 0.037 mg/L.
Therefore, 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.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL jars
- Type: Closed
- Material, size, headspace, fill volume: Glass, 50 mL, approximately 200 mL of test preparation
- Aeration: Not during the test, but the reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted tap water was used to prepare the Elendt M7 test medium
Typical water quality characteristics for the tap water as supplied, prior to reconstitution, are given below (taken from Goodband & Mullee 2012, Harlan Project No. 41103267).
- Total organic carbon: 0.64 to 1.39 mg/L (average 0.985 mg/L)
- Particulate matter: Not determined
- Metals [µg/L], average in brackets: Al < 5 to 52 (< 15.654), As < 0.37, Cd < 0.06 to 0.23 (< 0.121), Cr < 0.7 to 0.7 (< 0.7), Cu < 0.003 to 0.054 (< 0.015), Fe < 7 to 120 (< 20.808), Hg < 0.012, Mn < 1.5 to 11 (< 2.235), Ni 1.9 to 2.7 (2.288), Pb < 0.5 to 4.2 (< 1.35), Sb < 0.12 to 0.92 (< 0.329), Se < 0.22 to 1.1 (< 0.539)
- Pesticides: None above the LOQ (screening for numerous compounds)
- Chlorine: Free 0.01 to 0.46 mg/L (average 0.25 mg/L; Total 0.05 to 0.54 mg/L (average 0.312 mg/L)
- Conductivity: 269 to 487 µS/cm at 20 °C (average 358.885 µS/cm at 20 °C)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Last reporting period from 2009-01-01 to 2009-12-31

OTHER TEST CONDITIONS
- Adjustment of pH: The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl
- Photoperiod: 8 h dark / 16 h light with 20 min dawn and dusk transition periods

EFFECT PARAMETERS MEASURED
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 the daphnids were considered to be immobilised if they were unable to swim for approximately 15 s after gentle agitation.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: Nominal loading rates of 1.0, 10 and 100 mg/L were used in the range finding study.
- Results used to determine the conditions for the definitive study: No immobilization occurred.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (Harlan Laboratories Project No. 41104946) conducted between 29 November 2011 and 1 December 2011.

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: No immobilization in control and treatment replicates

Details on results:

- Behavioural abnormalities: No
- Mortality of control: No
- Other adverse effects control: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: The sole treatment Loading rate clearly exceeded the water solubility
- Vortex depth measurements: The height of the water column was 14.5 to 15.0 cm, the vortex depth was ca. 0.2 cm and the vortex was observed to be a dimple at the water surface on each occasion. The values were in agreement in the control and the treatment replicates and did not differ between start and end of the mixing period.

Results with reference substance (positive control):

The positive control used potassium dichromate at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. Analysis of the immobilisation data by geometric mean method at 24 h and the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 h based on the nominal test concentrations gave the following results: 24-h EC50 1.3 mg/L (95 % CL 1.0 to 1.8) and 48-h EC50 1.1 mg/L (95 % 1.0 to 1.3). The No Observed Effect Concentrations after 24 and 48 h were 1.0 and 0.56 mg/L respectively. The results were within the normal range for this reference item.
- Hamilton MA, Russo RC, Thurston RV (1977). Trimmed Spearman-Karber Method for Estimating Median Lethal Concentration in Toxicity Bioassays. Environ Sci Technol 11:714-19.

Validity criteria fulfilled:

yes

Conclusions:

Not acute toxic, no immobility and no difference to control up to enhanced water solubility (WAF), 48-h EL50 > 100 mg/L WAF, 48-h EL0 ≥ 100 mg/L WAF

Executive summary:

The short-term toxicity of the test item to aquatic invertebrates was investigated in a GLP-compliant study using daphnids (Daphnia magna) according to the EU C.2 (2008) and OECD TG 202 (1992) protocols. Appropriate Water Accommodated Fraction (WAF) to ensure exposure was prepared according to the OECD STA 23 (2000) and ECETOC Monograph no. 26 (1996) guidelines and generally accepted literature recommendations. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

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

No mortalities and no sub-lethal effects were observed in the treatment replicates and the control. Chemical analysis of the test preparations at 0 and 48 h showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method, which was determined to be 0.037 mg/L. Therefore, 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.

Accordingly the acute toxicity of the test item to the freshwater invertebrates gave a 48-h EL50 (Effective Loading 50 %) value of > 100 mg/L WAF. The 48-h EL0 was ≥ 100 mg/L WAF.

In conclusion the test item was found not acute toxic and no indication for aquatic hazard classification is given by the results of this study.

Description of key information

Not acute toxic, no immobility and no difference to control up to enhanced water solubility (WAF), 48-h EL50 > 100 mg/L WAF, 48-h EL0 ≥ 100 mg/L WAF (OECD 202)

Additional information

The short-term toxicity to aquatic invertebrates was investigated in a GLP-compliant study (Goodband & Mullee 2012, Harlan Report no. 41103268) using daphnids (Daphnia magna) according to the EU C.2 (2008) and OECD TG 202 (1992) protocols. Appropriate Water Accommodated Fraction (WAF) to ensure exposure was prepared according to the OECD STA 23 (2000) and ECETOC Monograph no. 26 (1996) guidelines and generally accepted literature recommendations. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions. No toxicity was found up to a loading rate of 100 mg/L WAF. Therefore it can be concluded that the submission item has no acute toxic effects on aquatic invertebrates up to the level of its enhanced water solubility.