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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 17 November 2011 and 13 January 2012

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

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Remarks:

see EU C.1 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 (incl. QA statement)

Remarks:

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

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 and 100 mg/L loading rate
- Sampling method: Duplicate samples were taken from both replicates at 0 h (fresh media), 24 h (old media), 72 h (fresh media) and 96 h (old media)
- Sample storage conditions before analysis: At approximately -20 °C

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Prior to use the test item was heated at approximately 50 °C to liquefy. An amount of test item (2100 mg) was added to the surface of 21 L of dechlorinated tap water to give the 100 mg/L loading rate. After the addition of the test item, the dechlorinated tap 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. Next step was 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 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 each mixing period and by observation of vortex for dimple at the water surface on each occasion.
- Evidence of undissolved material: Microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering. Microscopic observations of the WAF were performed after filtering and showed no particles of test item were present.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, U.K.
- Length at study end: Mean standard length 3.8 cm (SD ± 0.3)
- Weight at study end: Mean weight of 0.76 g (SD ± 0.13)
- Feeding during test: No

ACCLIMATION
- Acclimation period: The fish were in-house since 1 December 2011 (maintained in a glass fibre tank with a “single pass” water renewal system) and were acclimatised to test conditions from 28 December 2011 to 9 January 2012.
- Acclimation conditions: Same as test
- Type of food: Commercial trout pellets
- Feeding frequency: Daily; the stock fish were last fed approximately 3 h prior to the start of the definitive test.
- Health during acclimation (any mortality observed): No

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

Dilution water: Approximately 140 mg/L as CaCO3

Test temperature:

14 to 15 °C in control and sole treatment replicates, maintained at approximately 14 ºC in a temperature controlled room
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 Hanna Instruments HI 93510 digital thermometer.

pH:

7.9 to 8.5 in the control and 7.8 to 8.2 in the WAF treatment replicates (sole loading rate 100 mg/L); range in fresh media 7.8 to 8.1 versus 8.0 to 8.2 in old media; generally the pH in the fresh media in control and WAF were equal or max. 0.2 pH units lower in the WAF
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:

9.9 to 10.3 (control) and 9.8 to 10.3 (sole treatment replicates) mg/L
98 to 101 (control) and 95 to 100 (sole treatment replicates) % of Air Saturation Value
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.

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 samples the measured concentration was < LOQ. In the 100 mg/L loading 0.115 and 0.225 mg/L were fond at 0 h (fresh media) , 0.0637 and 0.0848 mg/L at 24 h (old media), 0.0190 and 0.0718 mg/L at 72 h (fresh media) and In summary the analysis of the fresh test preparations at 0 and 72 h showed measured concentrations ranging from 0.0190 to 0.225 mg/L and the analysis of the old media at 24 and 96 h showed measured concentrations ranging from less than the limit of quantitation (LOQ) of the analytical method employed (assessed as 0.019 mg/L) to 0.0848 mg/L.
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 (irrespective of the actual concentration of test item in the WAF).

Details on test conditions:

TEST SYSTEM
- Test vessel: Type: Closed, Material: Glass, Fill volume: 20 L
- Aeration: The test vessels were aerated via narrow bore glass tubes.
- Renewal rate of test solution: Daily renewal
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.27 g bw/L at test end

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given below.
- 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: No
- Photoperiod: 8 h dark / 16 h light with 20 min dawn and dusk transition periods

EFFECT PARAMETERS MEASURED
Any mortalities and sub-lethal effects of exposure were recorded at 3, 24, 48, 72 and 96 h after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: One nominal loading rate of 100 mg/L was used in the range finding study.
- Results used to determine the conditions for the definitive study: No mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: No mortality in control and treatment replicates

Duration:

96 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks on result:

other: No sub-lethal effects in control and treatment replicates

Details on results:

- Behavioural abnormalities: No
- Observations on body length and weight: No
- Mortality of control: 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 33.5 to 35.0 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 all four mixing periods in the control and the treatment replicates and did not differ between start and end of the mixing period.

Validity criteria fulfilled:

yes

Conclusions:

Not acute toxic, no mortality and no difference to control up to enhanced water solubility (WAF), 96-h LL50 and EL50 > 100 mg/L WAF, 96-h EL0 and LL0 ≥ 100 mg/L WAF

Executive summary:

The short-term toxicity of the test item to fish was investigated in a GLP-compliant study using rainbow trout (Oncorhynchus mykiss) according to the EU C.1 (2008) and OECD TG 203 (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 the fish were exposed, in two groups of seven, to a WAF of the test item, at a single nominal loading rate of 100 mg/L for a period of 96 h at a temperature of approximately 14 °C under semi-static test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 h after the start of exposure and then daily throughout the test until termination after 96 h.

No mortalities and no sub-lethal effects were observed in the treatment replicates and the control. The analysis of the fresh test preparations at 0 and 72 h showed measured concentrations to ranged from 0.0190 to 0.225 mg/L and the analysis of the old media at 24 and 96 h showed measured concentrations ranging from less than the limit of quantitation (LOQ) of the analytical method employed (assessed as 0.019 mg/L) to 0.0848 mg/L. 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 (irrespective of the actual concentration of test item in the WAF).

Accordingly the acute toxicity of the test item to freshwater fish gave 96-h LL50 (Lethal Loading 50 %) and EL50 (Effective Loading 50 %) values of > 100 mg/L WAF. The 96-h EL0 and LL0 were ≥ 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 mortality and no difference to control up to enhanced water solubility (WAF), 96-h LL50 and EL50 > 100 mg/L WAF, 96-h EL0 and LL0 ≥ 100 mg/L WAF (OECD 203).

Additional information

The short-term toxicity to fish was investigated in GLP-compliant study (Goodband & Mullee 2012, Harlan Report no. 41103267) using rainbow trout (Oncorhynchus mykiss) according to the EU C.1 (2008) and OECD TG 203 (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 fish up to the level of its enhanced water solubility.