2,2'-methylenebis(4,6-di-tert-butyl-phenyl)-2-ethylhexyl phosphite

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:

28 October 1999 and 4 February 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Sodium chloride (10g) was added to each of the aqueous samples (100 ml) before they were extracted by liquid-liquid partitioning into dichloromethane (2 x 20 ml). The organic extracts were evaporated to dryness and the residues dissolved in sufficient HPLC mobile phase to bring the expected analyte concentration within the calibration range; those at higher concentrations (1.05 to 121 mg/l) were dissolved in ethanol initially. The processed samples were analysed for ADK STAB HP-10 by high performance liquid chromatography using a spectrophotometric detector.

Vehicle:

yes

Details on test solutions:

Method of preparation: The test medium was prepared by adding the test substance (200 mg) mixed with tetrahydrofuran (0.2 ml) to algal nutrient medium (2 1). To aid dissolution and/or dispersion, this aqueous mixture was treated by ultrasound (twenty minutes) and then covered to exclude light and stirred (twenty-three hours) in the test area. The resultant aqueous mixture was then left to stand for approximately one hour before being filtered (cellulose nitrate filter, 0.2 μm pore size); it was then inoculated with algal cells (15 ml of the algal inoculum) to give an initial cell density of 1E04 cells/ml.

Stability of test concentration: The concentration of a saturated solution of ADK STAB HP-10 was measured using an HPLC method of chemical analysis. At the start of the definitive test, three samples (100 ml) were taken from additional flasks containing the freshly prepared control and test media; after 72 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. Additional samples were also taken from flasks containing ADK STAB HP-10 but with no algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells. The samples were stored in a freezer (-20°C) until two from each set were analysed; the other sample remained in storage in case further analysis was required. In order to verify the unexpectedly low concentration measured in samples taken at 72 hours, the stored sample was subsequently analysed.

Test organisms (species):

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

Details on test organisms:

Name: Selenastrum capricornutum, Strain No. CCAP 278/4.
Source: Axenic, uni-algal agar slope cultures of algae were obtained from the Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Cumbria, UK and arrived on 26 January 2000.
Pre-culture: The agar slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture and these primary liquid cultures (50 ml) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures which were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth (with a cell density of 6.31 E05 cells/ml). Aliquots of this culture were used in the study.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observatiob period specified in the study report.

Hardness:

Not specified in the study report

Test temperature:

23.3 to 24.0°C

pH:

7.0 - 8.2

Dissolved oxygen:

Not specified in the study report.

Salinity:

Not applicable - freshwater study

Nominal and measured concentrations:

Nominal concentrations of 0.1, 1, 10 and 100 mg/l.

Details on test conditions:

Experimental design and test concentrations: A range finding test was conducted at nominal test concentrations of 0.1, 1, 10 and 100 mg/l. The test media were prepared as described in 'Method of preparation' except that tetrahydrofuran was not employed and the test media were not filtered before use; no inhibition of growth occurred during the test.
Based on the results of this test, the definitive test employed a saturated solution of ADK STAB HP-1 0 prepared from an aqueous mixture with an initial nominal concentration of 100 mg/l. An algal nutrient medium control and a solvent control (0.1 ml tetrahydrofuran/l) were also established.
Before the start of the test, the required number of empty test vessels (250 ml conical flasks) were loosely stoppered with non-absorbent cotton wool, covered with aluminium foil which was secured by autoclave tape and sterilised by autoclaving (121°C for 15 minutes). Following the addition of inoculated test medium (100 ml), each flask was then loosely plugged with non-absorbent cotton wool.
The algal nutrient medium control and solvent control (0.1 ml tetrahydrofuran/l) cultures were prepared, without the addition of test substance, as described above for the test medium.

Environmental conditions: Conical flasks each containing 100 ml of control or test culture were placed in an illuminated orbital incubator according to a random number sequence. The cultures were incubated, without renewal of medium, for 72 hours under continuous illumination of approximately 8220 lux provided by 6 x 30 W "cool white" one metre fluorescent tubes. The temperature was maintained at 23.3 to 24.0°C.
The temperature and pH of control and test flasks at the start and end of the test were recorded. Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at a nominal 150 cycles per minute. The minimum, maximum and ambient temperature and light intensity in the test area were determined each day.

Measurement of Growth: Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities measured using a haemacytometer (Improved Neubauer); the cells were also examined for morphological abnormalities. The estimate of cell numbers in each sample was based on the mean of four or eight consecutive counts depending on the cell density of the cultures.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 19.3 µg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 19.3 µg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

biomass

Details on results:

Algal growth: Compared to the solvent control cultures, the area under the growth curve and the growth rate were reduced by 10% and 1% respectively at a mean measured ADK STAB HP-10 concentration of 19.3 μg/l.
Although statistically significant, this level of inhibition is not considered to be of biological significance.
The 72-hour median effect concentrations (EbC50 and ErC50) were not identified but they must be greater than 19.3 μg/l.
The "no-observed effect concentration" for inhibition of growth was ≥ 19.3 mg/l.

Observations: No microscopic abnormalities of the cells were detected.

Environmental parameters: The measurements of water quality (temperature and pH) in control and test flasks remained within acceptable limits throughout the study.
The aqueous mixture used to prepare the saturated solution was a non-homogeneous, off-white, hazy dispersion with particulate material visible on its surface and on the base of the preparation flask. The saturated solution was clear and colourless.

Results with reference substance (positive control):

Reference substance not used in this study.

Reported statistics and error estimates:

The "no-observed effect concentration" for inhibition of growth was ≥ 19.3 mg/l. Although the results of statistical analysis (Dunnetts Multicomparsion Test) for area under the growth curve showed a significant difference between the solvent control group and the test group it was not considered to be biologically significant as only a 10% reduction in growth was noted. Also, it was noted during the study that there was a slight stimulation of cell growth in the solvent control group compared to that in the nutrient medium control and the test group.

TABLE 2 – Cell densities and test cultures

ADK STAB HP-10 concentrations		Replicate number	Cell densities (x 10^4cells/ml)
Nominal (mg/l)	Measured (μg/l)		24 hours	48 hours	72 hours
Control	<LOQ	R1	8.000	21.00	100.5
		R2	7.250	24.50	106.0
		R3	7.625	22.88	104.0
		R4	6.500	22.00	106.3
		R5	7.375	21.50	106.3
		R6	7.000	19.75	103.0
		Mean	7.292	21.94	104.3
Solvent control	<LOQ	R1	6.000	30.50	136.3
		R2	9.875	31.50	113.5
		R3	9.250	30.50	114.0
		R4	9.500	29.50	108.5
		R5	11.000	29.50	114.0
		R6	8.375	31.00	117.3
		Mean	9.000	30.42	117.3
100 S	19.3	R1	8.875	30.13	112.0
		R2	6.500	28.25	105.8
		R3	8.125	30.88	120.0
		R4	8.500	30.50	99.00
		R5	8.125	31.50	119.8
		R6	6.750	27.00	106.8
		Mean	7.813	29.41	110.5

LOQ: limit of quantification (5.23 μg/l)

S: saturated solution

 

TABLE 3 – Inhibition of growth

ADK STAB HP-10 concentration		Replicate number	Area under curve (72h)^#	Mean (% Inhibition)	Growth rate (0-72h)^@	Mean (% Inhibition)
Nominal (mg/l)	Measured (μg/l)
Control	<LOQ	R1	1848	1894	6.410	6.455
		R2	1974		6.477
		R3	1921		6.451
		R4	1896		6.477
		R5	1905		6.477
		R6	1819		6.437
Solvent control	<LOQ	R1	2448	2294	6.823	6.615
		R2	2301		6.578
		R3	2262		6.578
		R4	2184		6.516
		R5	2280		6.578
		R6	2289		6.614
100 S	19.3	R1	1569	2060	6.553	6.534
		R2	2047	(10)	6.477	(1)
		R3	2317		6.649
		R4	2064		6.382
		R5	2331		6.649
		R6	2034		6.491

LOQ: limit of quantification (5.23 μg/l)

S: saturated solution

^#: x 10⁴

^@: x 10^-2

( ): % inhibition

 

TABLE 4 – Environmental parameters – temperature and pH

ADK STAB HP-10 concentration		Temperature°C		pH
Nominal (mg/l)	Measured (μg/l)	0h	72h	0h	72h
Control	<LOQ	22.3	23.2-23.6	7.6	7.0
Solvent control	<LOQ	22.4	23.1-23.8	8.0	7.3-7.5
100 S	19.3	22.7	22.8-23.6	8.2	7.3-7.4

LOQ: limit of quantification (5.23 μg/l)

S: saturated solution

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour EbC50 and ErC50 for a saturated solution of ADK STAB HP-10 were not identified but must be greater than 19.3 μg/l.
The "no-observed effect concentration" (NOEC) for inhibition of growth was ≥ 19.3 μg/l.

Executive summary:

The effect of ADK STAB HP-10 on the growth of the unicellular green alga Selenastrum capricornutum was assessed under non-axenic conditions.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC Part C, Method 3 "Algal Inhibition Test" and Procedure 201 of the OECD Guideline for Testing of Chemicals "Alga, Growth Inhibition Test".

Six replicate algal cultures, with an initial cell density of 1 x 10⁴/ml, were exposed to a saturated solution of ADK STAB HP-10 prepared in algal nutrient medium from an aqueous mixture at an initial nominal concentration of 100 mg/l. The saturated solution was prepared by adding the test substance to nutrient medium; to aid dissolution and/or dispersion, tetrahydrofuran and ultrasound treatment, followed by twenty three hours of stirring, were employed. The resultant mixture was filtered (cellulose nitrate filter, 0.2 μm pore size) before use in the test. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 23.3 to 24.0°C for 72 hours.

The measured concentration of ADK STAB HP-10 in samples of the test medium decreased during the test from 45.9 μg/l at the start to 8.09 μg/l (18% of its starting value) at 72 hours. The overall mean measured concentration of ADK STAB HP-10 was 19.3 μg/l. Although no explanation can be given for the loss of ADK STAB HP-10 from the test medium, it was not associated with the presence of algal cells since similar losses were observed in test medium incubated without algal cells.

Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate.

Compared to the solvent control cultures, the area under the growth curve and the growth rate were reduced by 10% and 1% respectively at a mean measured ADK STAB HP-10 concentration of 19.3 μg/l. Although statistically significant, this level of inhibition is not considered to be of biological significance.

The 72-hour median effect concentrations (EbC50and ErC50) were not identified but they must be greater than 19.3 μg/l.

The "no-observed effect concentration" for inhibition of growth was ≥19.3μg/l.

Description of key information

The 72 -hour median effect concentrations (EbC50and ErC50) were not identified but they must be greater than 19.3 μg/l. The "no-observed effect concentration" for inhibition of growth was ≥19.3μg/l.

The substance is not acutely toxic to aquatic organisms above the limit of water solubility, 0.59 mg/L. Surrogate PNEC of 0.59/100 = 0.0059 mg/L will be used.

Additional information

The effect of ADK STAB HP-10 on the growth of the unicellular green algaSelenastrum capricornutumwas assessed under non-axenic conditions.

Six replicate algal cultures, with an initial cell density of 1 x 10⁴/ml, were exposed to a saturated solution of ADK STAB HP-10 prepared in algal nutrient medium from an aqueous mixture at an initial nominal concentration of 100 mg/l. The saturated solution was prepared by adding the test substance to nutrient medium; to aid dissolution and/or dispersion, tetrahydrofuran and ultrasound treatment, followed by twenty three hours of stirring, were employed. The resultant mixture was filtered (cellulose nitrate filter, 0.2 μm pore size) before use in the test. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging from 23.3 to 24.0°C for 72 hours.

The measured concentration of ADK STAB HP-10 in samples of the test medium decreased during the test from 45.9 μg/l at the start to 8.09 μg/l (18% of its starting value) at 72 hours. The overall mean measured concentration of ADK STAB HP-10 was 19.3 μg/l. Although no explanation can be given for the loss of ADK STAB HP-10 from the test medium, it was not associated with the presence of algal cells since similar losses were observed in test medium incubated without algal cells.

Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate.

Compared to the solvent control cultures, the area under the growth curve and the growth rate were reduced by 10% and 1% respectively at a mean measured ADK STAB HP-10 concentration of 19.3 μg/l. Although statistically significant, this level of inhibition is not considered to be of biological significance.

The 72-hour median effect concentrations (EbC50and ErC50) were not identified but they must be greater than 19.3 μg/l.

The "no-observed effect concentration" for inhibition of growth was ≥19.3μg/l.