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

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:

21 May 1996 to 30 September 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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:

18 to 180 mg/l and 0.45 to 10 mg/l

Vehicle:

yes

Details on test solutions:

PREPARATION OF TEST SOLUTIONS
The standard test procedures required generation of test solutions which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface).
The water solubility of ADK STAB HP-10 in distilled water at 20.0 ± 0.5°C is determined to be< 0.59 mg/l, using the column elution method (NOTOX Project 155442). The partition coefficient (n-octanol/water) of ADK STAB HP-10 was determined to be > 1.6 x 106 (log Paw > 6.2) at room temperature (NOTOX Project 155453).
Due to the strong hydrophobic character of the test substance, a solvent other than water had to be used to prepare stock solutions which could be used for preparation of test solutions in test medium. According to data of the sponsor, ADK STAB HP-10 was soluble in tetra-hydrofuran (THF) at 685 g/l and in acetone at 18 g/l.
A pre-test was performed to examine the behaviour of the test substance in the test medium. A weighed amount of 20 mg was added to 200 ml test medium. Magnetic stirring and ultra sonication were applied, but the major part of the test substance remained floating at the surface of the test solution. Hence, direct addition of the test substance was not further considered. Instead, the following procedure was applied. A stock was prepared in THF and exact volumes were added to each test vessel. Then the THF was allowed to evaporate overnight before adding the test medium. After the volumes of test medium had been added to each vessel, these solutions were stirred for ca. 24 hours. After this period of stirring, thin parts of test substance were found floating at the surface of the test solutions. However, no test substance particles were found dispersed in the test solutions or at the bottom of the test vessels.
After evaluation of the responses recorded in the first EC50 test and the analytical data, it was decided to perform an additional test starting with stock solutions in acetone. Owing to the fact that the maximum solubility of ADK STAB HP-10 in acetone was 18 g/l, the highest nominal concentration should be 1.8 mg/l if the concentration of acetone was kept at 0.1 ml/l. Hence, it was decided to increase the concentration of acetone to 1 ml/l, so that a range of nominal concentrations > 1 mg/1 could be tested. A solvent control containing 1 ml/l acetone was included. Separate stocks in acetone were prepared and 0.25 ml of each stock was added to 250 ml test medium which was stirred during addition of the stock. Then the test solution was treated by ultra sonication for ca. 5 minutes to reach a complete dispersion of the test substance.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
Reason for selection: This system has been selected as an internationally accepted species.
Validity of batch: Frequent inspection of the cultures with respect to number of young, appearance of young and parental daphnia and global feeding behaviour.
Characteristics: For the test selection of young daphnia with an age of < 24 hours.
Start of each batch: With newborn animals, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C, constant within ± 1°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M4, as prescribed by Dr. Elendt-Schneider (Elendt, 8.-P., 1990: Selenium deficiency in
Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasms 154, 25-33).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

250 mg/l expressed as CaCO3

Test temperature:

18-22°C, constant within ± 1°C

pH:

8.0 ± 0.2 after aeration

Dissolved oxygen:

7.9 - 9.1 mg O2/l

Salinity:

No applicable - freshwater test

Nominal and measured concentrations:

nominal concentrations

Details on test conditions:

RANGE-FINDING TEST: A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Daphnia were exposed for 48 hours to test solutions containing nominal concentrations of 1, 10 and 100 mg/l based on the amounts of test substance dissolved in THF.

EC50 TESTS: TEST CONCENTRATIONS
First EC50 test: Based on the results of the range-finding test, a range of nominal concentrations from 18 to 180 mg/l, increasing by a factor of 1.8.
Second EC50 test: Nominal concentrations ranging from 0.45 to 10 mg/l, increasing by a factor of 2.2.
Controls: Test medium without test substance or other additives (0 mg/l), and one control containing test medium with the additives used in the treatment of the stock solutions.

EC50 TESTS: TEST PROCEDURE AND CONDITIONS
Test type: Static
Test duration: 48 hours
Test vessels: 100 ml, all-glass
Medium: ISO, prepared in milli-RO water
Number of daphnia: 20 per concentration
Loading: 10 per vessel containing 80 ml medium
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of young Daphnia: The daphnia were introduced into the test medium directly after preparation of the test solutions.

SAMPLING FOR ANALYSIS OF TEST CONCENTRATIONS
At the start and the end of the first EC50 test samples for analysis were taken from 18 and 180 mg/l. Also samples were taken from the controls. The samples were stored in a deep-freeze until analysis.
During the second EC50 test samples for analysis were taken as follows:

Frequency: From all test solutions at t=0h; From 4.5 and 10 mg/l at t=48h.
Volume: 10 ml from the approximate centre of the test vessel.
Storage: The samples were analysed on the day of sampling.

Additionally, reserve samples of 10 ml were taken from all concentrations. These samples were stored in a deep-freeze for possible analysis until delivery of the final report. Specification of the samples analysed and the method of analysis are described in the appended Analytical Report.

MEASUREMENTS AND RECORDINGS
Immobility (including mortality): At 24 hours and 48 hours.
pH and Oxygen: At the beginning and at the end of the test, for all concentrations and the control(s).
Temperature of medium: Daily in one control vessel, beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding test:The EC50 was expected to be > 100 mg/1 based on nominal concentrations.
Thin parts of test substance were observed floating at the surface of the treated test solutions.

First EC50 test: The responses recorded in this test allowed for reliable determination of an EC50 based on nominal concentrations. The 48h-EC50 was 51 mg/l (95% confidence interval between 39 and 74 mg/l). Hence, toxicity was higher than expected. This was probably related to a possible additional effect of THF residues still present in the test vessels after evaporation. Observation of the daphnia present in the treatment-control showed that these, although mobile, were not in optimal condition as compared to the untreated controls.
Analysis of samples taken from the lowest and the highest test concentration failed to show interpretable results. At the start of the test, a measured concentration of 1.9 mg/l (11 %) was found at nominal 18 mg/l, while a concentration of 0.2 mg/l was measured in the sample taken at 180 mg/l (<1 %). At the end of the 48-hour exposure period, 0.2 mg/l was measured in the sample taken at 18 mg/l, while no detectable concentration was found at nominal 180 mg/l. Hence, the responses recorded could not be related to actual exposure concentrations. Therefore, the effects recorded probably were caused by uptake of undissolved particles by the daphnia or by mechanical damage due to the presence of undissolved particles.

Second EC50 test: This test showed 45% immobilisation at the nominal concentration of 10 mg/l, while no effects on mobility were observed at the lower test concentrations. Analysis of the samples taken at the start of the test showed much higher recoveries of test substance (34 to 53 %) than found in the first test. The initial concentration was 4.6 mg/l at the nominal concentration of 10 mg/l and 2.4 mg/l at the nominal concentration of 4.5 mg/l. At the end of the 48-hour exposure period, the measured concentrations had decreased to 0.45 and 0.23 mg/l, respectively. These concentrations corresponded with levels approximating the water solubility of ADK STAB HP-10 being < 0.59 mg/l (NOTOX Project 155442).

Experimental conditions: The temperature of the test medium measured in the blank control varied within the range of 20 to 21 °C in both EC50 tests.

Results with reference substance (positive control):

All concentrations of ADK STAB HP-10 tested exceeded its solubility in water. The 48h-EC50 depended on the way the test solutions were prepared.
The sensitivity of the different batches of daphnia used were considered to be similar as the 48-hour EC50 was 0.6 mg/l in both reference tests.

Table 1: Incidence of immobility in the range-finding test:

Concentration Test substance (mg/l)	Number Daphnia exposed	Response at 24h		Response at 48h
		Number	%	Number	%
Blank-contr.	10	0	0	0	0
Additive-cont.	10	0	0	0	0
1.0	10	0*	0	0*	0
10	10	0*	0	0*	0
100	10	0*	0	4*	40

*Thin parts of substance were observed at the surface of the test solution.

 

Table 2: Acute immobilization of daphnia after 24 and 48 hours in the first EC50-test with various concentrations of ADK STAB HP-10

Concentration substance (mg/l)		Vessel code	Number exposed	Number immobile at 24h	Number immobile at 48h
Nominal	Actual
Blank-control		A	10	0	0
		B	10	0	0
Treatment-control		A	10	0	0
		B	10	0	0
18	1.9 - 0.2	A	10	0*	0*
		B	10	0*	0*
32	n.m.	A	10	0*	1*
		B	10	0*	6*
56	n.m.	A	10	0*	3*
		B	10	0*	5*
100	n.m.	A	10	2*	10*
		B	10	1*	8*
180	0.2	A	10	9*	10*
		B	10	10*	10*

*Thin scales of test substance were observed at the surface of the solution.

n.m. = Not measured by analysis of samples.

 

Table 3: Acute immobilization of daphnia after 24 and 48 hours in the second EC50-test with various concentrations of ADK STAB HP-10

Concentration (mg/l)		Vessel code	Number exposed	Number immobile at 24h	Number immobile at 48h
Nominal	Actual^1
Blank-control		A	10	0	0
		B	10	0	0
Treatment-control		A	10	0	0
		B	10	0	0
0.45	n.d.	A	10	0	0
		B	10	0	0
1.0	0.36	A	10	0	0
		B	10	0	0
2.2	0.75	A	10	0	0
		B	10	0	0
4.5	2.4	A	10	0*	0*
		B	10	0*	0*
10	4.6	A	10	0*	4*
		B	10	0*	5*

¹Initial concentrations measured in samples taken at the start of the exposure.

*Thin scales of the test substance were observed at the surface of the solution.

n.d. = not detected. 

Validity criteria fulfilled:

yes

Conclusions:

Nominal concentrations of 4.5 mg/1 and lower did not induce acute immobilization of Daphnia magna after 48 hours of exposure (NOEC).

Executive summary:

Acute Toxicity study in Daphnia magna with ADK STAB HP-10.

The study procedures described in this report were based on the EEC directive 92/69, Part C.2. "Acute toxicity for Daphnia" and the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 4, 1984,

The water solubility of ADK STAB HP-10 in distilled water at 20.0±0.5°C is determined to be< 0.59 mg/l, using the column elution method (NOTOX Project 155442). The partition coefficiant (n-octanol/water) of ADK STAB HP-10 was determined to be>1.6 x 10⁶(log Pow>6.2) at room temperature (NOTOX Project 155453).

Daphnia were exposed for a maximum of 48 hours in two separate tests to a range of nominal concentrations from 18 to 180 mg/l and 0.45 to 10 mg/l, respectively. The test solutions in the first test were prepared starting with stocks in tetra-hydrofuran (THF) and those in the second test were prepared with stocks in acetone. Both tests were performed in duplicate with 10 daphnia per vessel.

All concentrations of ADK STAB HP-10 tested exceeded its solubility in water. The 48h-EC50 depended on the way the test solutions were prepared. The sensitivity of the different batches of daphnia used were considered to be similar as the 48-hour EC50 was 0.6mg/l in the corresponding reference tests with potassium dichromate.

When test medium was exposed to ADK STAB HP-10 concentrations present in test vessels after evaporation of the so1vent THF, the nominal 48h-EC50 corresponded with 51 mg/l (95% confidence interval between 39 and 74 mg/l). However, the analysis of samples did not provide reliable information about the corresponding actual exposure concentrations. Further, the effect on mobility at the higher nominal concentrations was probably related to the presence of undissolved test substance particles.

When test medium was mixed with volumes of ADK STAB HP-10 stocks in acetone, the 48h-EC50 approximated a nominal concentration of 10 mg/l. Nominal concentrations of 4.5 mg/l and lower did not induce acute immobilization of Daphnia magna after 48 hours of exposure (NOEC). The actual exposure concentrations decreased substantially during the 48-hour test period owing to the fact that this highly hydrophobic test substance precipitated out of the water phase.

In conclusion, these results showed that ADK STAB HP-10 was not toxic for Daphnia magna at levels beyond its water solubility (< 0.59 mg/l), except in those cases where initial concentrations measured exceeded 2.4 mg/l, and the fraction of undissolved test substahce particles reached amounts that most likely induced physical damage to Daphnia magna.

Description of key information

Key value determined using EU Method C2 and OECD guideline 202.

Additional information

The water solubility of ADK STAB HP-10 in distilled water at 20.0±0.5°C is determined to be< 0.59 mg/l, using the column elution method (NOTOX Project 155442). The partition coefficiant (n-octanol/water) of ADK STAB HP-10 was determined to be>1.6 x 10⁶(log Pow>6.2) at room temperature (NOTOX Project 155453).

Daphnia were exposed for a maximum of 48 hours in two separate tests to a range of nominal concentrations from 18 to 180 mg/l and 0.45 to 10 mg/l, respectively. The test solutions in the first test were prepared starting with stocks in tetra-hydrofuran (THF) and those in the second test were prepared with stocks in acetone. Both tests were performed in duplicate with 10 daphnia per vessel.

All concentrations of ADK STAB HP-10 tested exceeded its solubility in water. The 48h-EC50 depended on the way the test solutions were prepared. The sensitivity of the different batches of daphnia used were considered to be similar as the 48-hour EC50 was 0.6mg/l in the corresponding reference tests with potassium dichromate.

When test medium was exposed to ADK STAB HP-10 concentrations present in test vessels after evaporation of the so1vent THF, the nominal 48h-EC50 corresponded with 51 mg/l (95% confidence interval between 39 and 74 mg/l). However, the analysis of samples did not provide reliable information about the corresponding actual exposure concentrations. Further, the effect on mobility at the higher nominal concentrations was probably related to the presence of undissolved test substance particles.

When test medium was mixed with volumes of ADK STAB HP-10 stocks in acetone, the 48h-EC50 approximated a nominal concentration of 10 mg/l. Nominal concentrations of 4.5 mg/l and lower did not induce acute immobilization of Daphnia magna after 48 hours of exposure (NOEC). The actual exposure concentrations decreased substantially during the 48-hour test period owing to the fact that this highly hydrophobic test substance precipitated out of the water phase.

In conclusion, these results showed that ADK STAB HP-10 was not toxic for Daphnia magna at levels beyond its water solubility (< 0.59 mg/l), except in those cases where initial concentrations measured exceeded 2.4 mg/l, and the fraction of undissolved test substahce particles reached amounts that most likely induced physical damage to Daphnia magna.