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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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.3 (Algal Inhibition test)

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 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Remarks:

see EU C.3 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 (replicates R1 - R6 pooled) and 100 mg/L loading rate (replicates R1 - R6 pooled)
- Sampling method: Duplicate samples were taken at 0 and 72 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 culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium 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 2 h. Next the WAF was filtered 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. An aliquot (1 L) of the WAF was inoculated with algal suspension (12.2 mL) to give the required test concentration of 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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: CCAP 278/4
- Source: Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Ports (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland, U.K. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.

ACCLIMATION
- Acclimation period: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
- Culturing media and conditions: Same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test. The temperature within the incubator was recorded daily.

pH:

8.1 to 8.3 (control at 0 and 72 h, respectively) and 7.9 to 8.2 (sole treatment replicates at 0 and 72 h, respectively)
The pH deviation in the control cultures was less than 1.5 pH units after 72 h and therefore was within the limits given in the Test Guidelines.
The pH of each control and test flask was determined at initiation of the test and after 72 h exposure using a WTW pH 320 pH 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: At test start (0 h) 0.0692 mg/L and at test end (72 h) < LOQ (i.e. 0.014 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: Conical flasks
- Type: Closed; flasks plugged with polyurethane foam bung
- Material, size, headspace, fill volume: Glass, 250 mL, 150 mL, 100 mL
- Initial cells density: Mean 5070 cells/mL (Pre-culture conditions gave an algal suspension in Log phase growth characterised by a cell density of 4.09 ∙ 10^5 cells per mL. 1 L of test medium was inoculated with 12.2 mL of this algal suspension to give approximately 5000 cells/mL .)
- Control end cells density: Mean 861,000 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+). Typical water quality characteristics for the tap water as supplied, prior to reverse osmosis, 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
- Sterile test conditions: No
- Adjustment of pH: To 7.5 ± 0.1 with 0.1 N NaOH or HCI
- Photoperiod: Continuous illumination provided by warm white lighting (380 - 730 nm)
- Light intensity and quality: Approximately 7000 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 h and the cell densities determined using a Coulter® Multisizer Particle Counter.
- All test and control cultures were inspected microscopically at 72 h.

TEST CONCENTRATIONS
- Test concentrations: Nominal loading rates of 10 and 100 mg/L were used in the range finding study.
- Results used to determine the conditions for the definitive study: No effect on growth at 10 and 100 mg/L loading rate WAF occurred.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (Harlan Laboratories Project No. 41104038) conducted between 10 October 2011 and 13 October 2011

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Slight hormesis rather than inhibition compared to control in growth rate (mean 1 %) and yield (mean 6 %)

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test cultures.
- Any stimulation of growth found in any treatment: Slight hormesis of 1 % (growth rate) and 6 % yield was observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-h test period all control and test cultures were observed to be pale green dispersions.
- 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 15 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 concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0-72 h) 1.4 mg/L (95 % CL 1.2–1.7 mg/L), NOErC 0.25 mg/L, LOErC 0.50 mg/L
EyC50 (0–72 h) 0.59 mg/L (95 % CL 0.53 – 0.65 mg/L), NOEyC 0.25 mg/L, LOEyC 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Statistical analysis of the growth rate and yield data was carried out for the control and 100 mg/L loading rate WAF test group after 72 h using a Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences (p ≥ 0.05), between the control and 100 mg/L loading rate WAF test group and therefore the “Effective Loading” rate for 0 % (EL0) based on growth rate and yield was equal to or greater than 100 mg/L WAF.
- SAS/STAT Proprietary Software Release 8.02 (1999 - 2001), SAS Institute Inc, Cary, NC, U.S.A.
- Sokal RR, Rohlf FJ (1981). Biometry. New York: W H Freeman and Company.

Test item stability investigations

At test start (0 h) 0.0692 mg/L and at test end (72 h) < LOQ of the analytical method employed, which was determined to be 0.014 mg/L, were measured in the treatments. 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.

In the cell-free additional sample of the 100 mg/L WAF a measured concentration of less than the LOQ was obtained indicating that the decline observed was due to instability rather than adsorption of the test item to the algal cells present. Nonetheless another possible explanation would be sorption to materials of the test system.

Table 2: Cell Densities and in the Definitive Test

Nominal Loading Rate [mg/L]		pH	Cell Densities* [cells per mL]				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.1	5.06E+03	2.20E+04	1.58E+05	8.56E+05	8.3
	R2		5.01E+03	3.49E+04	2.00E+05	1.07E+06
	R3		5.06E+03	2.69E+04	1.30E+05	6.17E+05
	R4		5.08E+03	2.57E+04	1.57E+05	8.57E+05
	R5		5.07E+03	3.27E+04	1.59E+05	7.62E+05
	R6		5.11E+03	2.94E+04	1.66E+05	1.01E+06
	Mean		5.07E+03	2.86E+04	1.62E+05	8.61E+05
100	R1	7.9	4.92E+03	3.22E+04	1.54E+05	1.02E+06	8.2
	R2		4.35E+03	3.43E+04	2.06E+05	9.67E+05
	R3		4.94E+03	2.44E+04	1.31E+05	6.92E+05
	R4		5.16E+03	3.16E+04	2.01E+05	1.14E+06
	R5		4.55E+03	3.08E+04	1.82E+05	9.23E+05
	R6		4.95E+03	2.43E+04	1.38E+05	7.08E+05
	Mean		4.81E+03	2.96E+04	1.69E+05	9.09E+05

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 to R6= Replicates 1 to 6

Table 3: Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate [cells/mL/h]
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.062	0.082	0.070
	R2	0.081	0.073	0.070
	R3	0.070	0.066	0.065
	R4	0.068	0.075	0.071
	R5	0.078	0.066	0.065
	R6	0.074	0.072	0.075
	Mean	0.072	0.072	0.069

R1 to R6 = Replicates 1 to 6

Table 4: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate [mg/L]		Growth Rate [cells/mL/h]		Yield [cells/mL]
		0 - 72 h	% Inhibition	0 - 72 h	% Inhibition*
Control	R1	0.071	not applicable	8.51E+05	not applicable
	R2	0.074		1.06E+06
	R3	0.067		6.12E+05
	R4	0.071		8.52E+05
	R5	0.070		7.56E+05
	R6	0.074		1.00E+06
	Mean	0.071		8.56E+05
	SD	0.003		1.63E+05
100	R1	0.074	-4	1.02E+06
	R2	0.073	-3	9.62E+05
	R3	0.068	4	6.87E+05
	R4	0.075	-6	1.14E+06
	R5	0.072	-1	9.18E+05
	R6	0.069	3	7.03E+05
	Mean	0.072	-1	9.04E+05	-6
	SD	0.003	not determined	1.77E+05	not determined

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated.

R1 to R6= Replicates 1 to 6

SD= Standard Deviation

 

Validity criteria fulfilled:

yes

Conclusions:

Not toxic, no difference to control up to enhanced water solubility (WAF), 72-h EL50 > 100 mg/L WAF, 72-h EL0 ≥ 100 mg/L WAF

Executive summary:

The toxicity of the test item to aquatic algae was investigated in a GLP-compliant study using freshwater green algae (Pseudokirchneriella subcapitata) according to the EU C.3 (2008) and OECD TG 201 (2006) 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 test organisms were exposed to a WAF of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) for 72 h, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Analysis of the 100 mg/L loading rate WAF test preparation at 0 h showed a measured test concentration of 0.069 mg/L. A decline in measured test concentration was observed at 72 h to less than the limit of quantitation (LOQ) of the analytical method, which was determined to be 0.014 mg/L. An additional sample of the 100 mg/L loading rate WAF, prepared with the omission of algal cells was incubated alongside the test from which a sample was taken for chemical analysis at 72 h. A measured concentration of less than the LOQ was obtained indicating that the decline observed was due to instability or sorption to the test system rather than adsorption of the test item to the algal cells present. Given that the 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.

Exposure to the test item resulted in a minor hormesis effect in growth rate (mean 1 %) and yield (mean 6 %). Accordingly the acute toxicity of the test item to the freshwater algae gave a 72-h EL50 (Effective Loading 50 %) value of > 100 mg/L WAF. The EL0 was ≥ 100 mg/L WAF.

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

Description of key information

Not toxic, no difference to control up to enhanced water solubility (WAF), 72-h EL50 > 100 mg/L WAF, 72-h EL0 ≥ 100 mg/L WAF (OECD 201)

Additional information

The toxicity to aquatic algae was investigated in a GLP-compliant study (Vryenhoef & Mullee 2011, Harlan Report no. 41103269) using freshwater green algae (Pseudokirchneriella subcapitata) according to the EU C.3 (2008) and OECD TG 201 (2006) 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 algae up to the level of its enhanced water solubility.