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

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:

18 November 1999 to 13 March 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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:

The mean measured concentration of ADK STAB HP-1 0 in samples of test medium ranged between 32.3 μg/l and 57.5 μg/l during the test, with an overall mean measured level of 42.6 μg/l.

Vehicle:

yes

Details on test solutions:

On each occasion, the test substance (1.4 g) mixed with tetrahydrofuran (1.4 ml) was added to diluents water (1.5 l) in a volumetric flask (2 l). To aid dissolution and/or dispersion, the contents of the flask were treated with ultrasound for twenty minutes before being made up to volume (2 l) and poured into a glass aquarium where the volume was adjusted to 14 litres with diluent water. The medium was then stirred for approximately twenty-four hours in the dark and subsequently left to stand in the test area for at least an hour; the resultant mixture was passed through a nylon cartridge filter (pore size 0.2 μm) before being used in the test.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Name: Rainbow trout (Oncorhynchus mykiss).
Source: The fish were supplied by Donnington Fish Farm, Gloucestershire, England. They were reared at the farm from English eggs which hatched in early September 1999.
Acclimatisation: The fish were delivered to the Huntingdon Research Centre, Cambridgeshire by the supplier on 21 December 1999. They were held outside in a supply of diluent water under flow-through conditions before being transferred by road on 5 January 2000 to the Eye Research Centre, where they were held in an aerated supply of diluent water under flow-through conditions until use. During the 14-day period immediately before the definitive test, water temperatures remained within the range 13.7 to 14.0°C, pH values within the range 7.7 to 7.8, dissolved oxygen concentrations within the range 87 to 93% air saturation value (ASV) and total hardness within the range 176 to 200 mg/l as CaCO3.
The fish were fed daily with commercial fish food (TROUW (UK) Ltd; Nutra Fry AB 02) an amount equivalent to 5% of the total wet-weight of fish in the holding tank. No food was given during the 21-hour period immediately before exposure or during the exposure period itself. No medication was given during the holding period, and mortalities were recorded as 4.4% in the 14 days before the definitive test.
The size of the fish used in the definitive study was determined by weighing and measuring a sample of ten fish taken from a sub-sample of the holding tank on 24 January 2000; their mean fork length was 4.0 cm and their mean wet weight was 1.2 g.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Total hardness within the range 176 to 200 mg/l as CaCO3.

Test temperature:

Water temperatures remained within the range 13.7 to 14.0°C

pH:

pH values within the range 7.7 to 7.8

Dissolved oxygen:

Dissolved oxygen concentrations within the range 87 to 93% air saturation value (ASV).

Salinity:

Not applicable

Nominal and measured concentrations:

The mean measured concentration of ADK STAB HP-10 in samples of test medium ranged between 32.3 μg/l and 57.5 μg/l during the test, with an overall mean measured level of 42.6 μg/l.

Details on test conditions:

DILUENT WATER: The water used to hold the fish and for the study was laboratory tap water, dechlorinated and softened by passage through an Elga® water purification system. It was passed through a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passed through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water were then remixed to give a supply with the desired water hardness. This water was then held in an intermediate tank where it was equilibrated to the test temperature and gently aerated before being supplied to the holding and test areas.

EXPOSURE CONDITIONS
Experimental design and test concentrations: A range finding test was conducted at nominal test concentrations of 1, 10 and 100 mg/l. The media were prepared as described above except that they were not filtered before the fish were added. No mortality occurred at 1 and 100 mg/l but one fish died (33%-mortality) at 10 mg/l. Based on the results of this test, the definitive test employed a saturated solution of ADK STAB HP-10 prepared from an aqueous mixture with a nominal concentration of 100 mg/l. A diluent water control and a solvent control (0.1 ml tetrahydrofuran/l) were also established. However, this test was not successful on the first occasion because a number of adverse symptoms were noted in all exposure groups and one fish died in the test vessel; these symptoms were probably caused by the presence of aggressive fish in all vessels. The test was repeated using a different batch of fish and the results obtained are reported here.
Ten fish were placed at random into each glass aquarium containing the prepared test or control media. Each vessel contained fourteen litres of medium to a depth of 19 cm. This provided an initial static loading of 0.86 g bodyweight/litre.
Medium renewal: The fish were exposed to the control or test conditions for a period of 96 hours with daily batch wise renewal of the media to ensure the maintenance of satisfactory environmental conditions and to maintain stable exposure levels.
Environmental conditions: The treatment and control groups were maintained at 13-15°C throughout the exposure period and constant to within ±1 °C during the study. The temperature of the water in the diluent control vessel was continuously monitored during the study.
Supplementary aeration was provided via narrow bore glass tubes. A photoperiod of 16 hours light:8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. Daily records of temperature, pH and dissolved oxygen were kept for the control and test vessel together with measurements of total hardness at 0 hours. The fish were not fed during the 96-hour exposure period.

CRITERIA OF EFFECT: The criteria of death employed in this study were (i) absence of respiratory movement and (ii) absence of response to physical stimulation of the caudal peduncle.
In addition to observations on mortality at approximately 15 minutes, 2, 4, 24, 48, 72 and 96 hours, subjective assessments were also made on the incidence and type of any sub-lethal effects compared with control fish.

EVALUATION OF DATA: The "no-observed effect concentration" (NOEC) was derived by direct inspection of the levels at which lethal and treatment-related-effects were observed during the test. An incidence rate of more than one affected fish out of ten is considered to be significant.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 42.6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 42.6 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Chemical analysis: In view of the low limit of aqueous solubility of ADK STAB HP-1 0 (<0.59 mg/l at 20.0 ± 0.5°C), the low concentration measured during the study was not unexpected. At the start of the test, the mean measured concentration of ADK STAB HP-10 in the test medium was 32.3 μg/l; at 24 hours, a mean concentration of 41.6 μg/l was measured in the expired medium. The mean concentration measured in freshly-prepared medium at 72 hours was below the limit of quantification of the analytical method (5.23 μg/l). Although this anomalous result cannot be explained, the mean concentration of ADK STAB HP-10 in the expired medium at 96 hours (57.5 μg/l) was similar to that found at 24 hours suggesting that the test medium had been correctly prepared at 72 hours. The overall mean measured concentration of ADK STAB HP-10 was 42.6 μg/l.

Mortality and observations: No mortalities or significant sub-lethal effects on the fish were noted during the test.
The 96-hour LC50 was not identified but must be >42.6 μg/l and the "no-observed effect concentration" (NOEC) was ≥ 42.6 μg/l.

Environmental parameters: The measurements of water quality (temperature, pH, concentrations of dissolved oxygen and total hardness) remained within acceptable limits throughout the study.
The aqueous mixture used to prepare the saturated solution was an off-white, hazy dispersion with particulate material visible on its surface and on the base of the vessel. The saturated solution was clear and colourless.

Results with reference substance (positive control):

Reference substance not used in this study.

Sublethal observations / clinical signs:

TABLE 1 – Measured concentrations

Nominal conc. mg/l	Measured ADK STAB HP-10 concentrations (μg/l)									Overall mean^#(μg/l)
	0 hours		24 hours		% ti	72 hours		96 hours
0^$	<LOQ	<LOQ	<LOQ	<LOQ	-	<LOQ	<LOQ	<LOQ	<LOQ	-
100 S	33.8	30.9	44.7	38.8	129	<LOQ*	<LOQ*	51.6	64.0	42.6
	[0.03]		[0.04]			-		[0.06]		(0.04)

LOQ = limit of quantification (5.23 μg/l)

% ti = mean measured concentration after 24 hours expressed as a percentage of the mean starting concentration (0 hours)

[ ] = mean measured concentration expressed as a percentage of the nominal concentration

( ) = overall mean expressed as a percentage of the nominal concentration

S = saturated solution

^$= both dilution water and solvent (0.1 ml tetrahydrofuran/l) controls

^#= geometic mean

* = the actual values measured (i.e. 0.487 to 1.21 μg/l) have not been included in the calculation of the overall mean

 

TABLE 2 – Environmental parameters; temperature, pH, dissolved oxygen and total hardness

ADK STAB HP-10 concentration		Temperature°C		pH		Dissolved oxygen (% ASV)		Total hardness mg/l as CaCO3
Nominal, mg/l	Measured,μg/l	Min	Max	Min	Max	Min	Max	0 hours
Control	<LOQ	13.4	14.8	7.6	8.2	80	98	176
Solvent control	<LOQ	13.3	14.9	7.6	8.3	78	98	-
100 S	42.6	13.7	14.9	7.7	8.3	82	97	-

ASV = air saturation value

LOQ = limit of quantification (5.23 μg/l)

S = saturated solution

Continuous monitoring of diluent control vessel media temperature = 13.3 to 15.2°C

Validity criteria fulfilled:

yes

Conclusions:

The 96-hour LC50 of a saturated solution of ADK STAB HP-10 was not identified but must be >42.6 μg/L.
The "no-observed effect concentration" was ≥42.6 μg/l.

Executive summary:

A study was performed to assess the acute toxicity of ADK STAB HP-10 to the rainbow trout (Oncorhynchus my kiss) under semi-static exposure conditions.

The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. No. L383A, 29.12.92) Part C, Method 1 "Acute Toxicity for Fish" and Procedure 203 of the OECD Guidelines for Testing of Chemicals, "Fish, Acute Toxicity Test".

A group of ten juvenile fish was exposed to a saturated solution of ADK STAB HP-10 prepared from an aqueous mixture with an initial nominal concentration of 100 mg/l. The saturated solution was prepared daily by adding the test substance to diluent water; to aid dissolution and/or dispersion, tetrahydrofuran and ultrasound treatment, followed by approximately twenty-four hours stirring, were employed. The resultant mixture was filtered (0.2 μm pore size, nylon filter) before use in the test.

The mean measured concentration of ADK STAB HP-10 in samples of test medium ranged between 32.3 μg/l and 57.5 μg/l during the test, with an overall mean measured level of 42.6 μg/l. This excludes the low result (<5 .23 μg/l) obtained in a sample of freshly-prepared medium; although this anomalous result cannot be explained, the mean concentration of ADK STAB HP-10 measured in samples of expired medium at 96 hours (57.5 μg/l) suggests that the test medium was correctly prepared at 72 hours.

Observations of the fish were made after approximately 0.25, 2, 4, 24, 48, 72 and 96 hours of exposure. No mortalities or treatment-related effects on fish were noted.

 

The 96-hour LC50 of a saturated solution of ADK STAB HP-10 was not identified but must be >42.6 μg/L.

The "no-observed effect concentration" was ≥42.6μg/l.

Description of key information

Key value determined using EU Method C1 and OECD guideline 203.

Additional information

A study was performed to assess the acute toxicity of ADK STAB HP-10 to the rainbow trout (Oncorhynchus my kiss) under semi-static exposure conditions.

A group of ten juvenile fish was exposed to a saturated solution of ADK STAB HP-10 prepared from an aqueous mixture with an initial nominal concentration of 100 mg/l. The saturated solution was prepared daily by adding the test substance to diluent water; to aid dissolution and/or dispersion, tetrahydrofuran and ultrasound treatment, followed by approximately twenty-four hours stirring, were employed. The resultant mixture was filtered (0.2 μm pore size, nylon filter) before use in the test.

The mean measured concentration of ADK STAB HP-10 in samples of test medium ranged between 32.3 μg/l and 57.5 μg/l during the test, with an overall mean measured level of 42.6 μg/l. This excludes the low result (<5 .23 μg/l) obtained in a sample of freshly-prepared medium; although this anomalous result cannot be explained, the mean concentration of ADK STAB HP-10 measured in samples of expired medium at 96 hours (57.5 μg/l) suggests that the test medium was correctly prepared at 72 hours.

Observations of the fish were made after approximately 0.25, 2, 4, 24, 48, 72 and 96 hours of exposure. No mortalities or treatment-related effects on fish were noted.

 

The 96-hour LC50 of a saturated solution of ADK STAB HP-10 was not identified but must be >42.6 μg/L.

The "no-observed effect concentration" was ≥42.6μg/l.