bis(2-ethylhexyl) (4-hydroxy-3,5-dimethoxybenzyl)malonate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-06-10 to 2009-02-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted 21 September 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

2001

Deviations:

no

Principles of method if other than guideline:

none

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentrations:
- Sampling method: For determination of the test item concentration, duplicate samples were taken from the freshly prepared test medium and from the control at one treatment period of the first, second and last week (Day 0, 7 and 16, respectively). To determine the maintenance of the test item concentration in the test medium, stability samples were taken at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19). Treatment samples and control samples were thawed at room temperature for two hours and shaken manually to obtain homogeneous sample solutions. Aliquots of the sample solutions were analyzed by LC-MS/MS.
- Sample storage conditions before analysis: Immediately after the samples were taken, an appropriate amount of methanol was added to each test medium sample (at the ratio 1:1) to stabilize the test item during the storage period. Then the samples were stored deep-frozen (at about –20 °C) until the analyses were performed. According to pre-experiments (non-GLP) to the storage stability the test item is sufficiently stable in test
media samples under these storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Test water: The test was conducted in reconstituted freshwater (Elendt M7 medium). Before use, the test water was aerated until oxygen saturation. During the test, the test media were not aerated. The test water was prepared by dissolving analytical grade salts and additives in purified water (see addendum).

- Method:
Due to the low solubility of the test item in the test water, a dispersion with the loading rate of 100 mg/L was prepared by dispersing 300 mg of the test item (effective weights: 292.02-309.6 mg) in 3000 mL of test water (effective volumes: 2920-3096 mL) using intense stirring. No auxiliary solvent or emulsifier was used. The dispersion was stirred with a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum amount of the test item in the dispersion.
The long stirring period of 96 hours was selected according to the results of a pre-experiment (non-GLP) which showed that the maximum concentration of test item was reached after a stirring period of this duration.
Following the stirring period of 96 hours, the test medium was incubated in a separating funnel for an equilibration period of 24 hours in the dark. During this period, different phases (a layer of test water with a maximum amount of dissolved test item and layers of undissolved test item at the surface, at the bottom and at the glass wall of the separating funnel) were observable and were separately let out through the stopcock of the separating funnel.
Thereafter, the part of the dispersion with the maximum of dissolved test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm). The negative pressure of the filtration unit was reduced as far as possible to avoid losses of volatile components of the test item during filtration. The filtrate with the loading rate of 100 mg/L was used as the single concentrated test medium.
The test medium was prepared just before introduction of the daphnids (i.e., start of the test and prior to each test medium renewal).
The test medium preparation was performed as far as possible in the dark.
The preparation of the stock- and test solutions was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with females of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992. Since this date, the clone is successfully bred in culture medium identical to the medium used for the test. The temperature and light conditions were identical to those of the test.
The stock animals were maintained in one liter glass beakers filled with culture medium and were transferred twice a week to fresh medium. The animals were fed normally three times a week with green algae of the species Scenedesmus subspicatus and with a fish food suspension.
The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.
The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old.
The test method and test species, Daphnia magna, are recommended by the test guidelines.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

250 mg CaCO3

Test temperature:

19-21°C

pH:

7.6-8.0

Dissolved oxygen:

at least 7.8 mg/L

Nominal and measured concentrations:

nominal: 100 mg/L
measured: 0.1 µg/l

Details on test conditions:

Experimental Conditions
This semi-static test with test medium renewals every 48 or 72 hours was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 19-21 °C. A 16-hour light to 8-hour dark cycle with a 30 minute transition period was used. Light intensity during the light period was between approximately 500 to 630 Lux. In this semi-static test, the test media of all treatments were renewed on Days 2, 5, 7, 9, 12, 14, 16, and 19 of the test period (every Monday, Wednesday, and Friday). At these dates, the surviving test animals were carefully transferred by means of glass tubes from the old test vessels into the freshly prepared test medium. The test animals were fed daily (with the exeption of Day 3, when daphnids were not fed) with a food mixture containing a suspension of green algae of the species Scenedesmus subspicatus and a fish food suspension. The fish food suspension was prepared by dispersing the amount of 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, 49324 Melle / Germany) in 500 mL of test water. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with test water and boiled. The suspension was stored deep frozen in small quantities until use. The carbon contents of the algal and fish food suspensions were determined using a Shimadzu TOC 5000A Analyzer. The food amount (based on the measured concentrations of the total organic carbon (TOC) in the food suspensions) was 0.20 mg TOC per Daphnia and day.

Study Design
The study was started with 20 daphnids per treatment. Each test animal was kept individually in a glass beaker. The test animals were randomly distributed to the test vessels. The test vessels were covered with glass plates and were labeled with the study number and all necessary additional information to ensure unique identification. The test duration was 21 days. A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to its solubility limit in the test water. Thus, a single loading rate of the test item of 100 mg/L was tested. Additionally, a control was tested in parallel (test water without addition of the test item). The actual concentrations of the test item in the test medium were analytically determined. The limit test was based on the results of range finding tests (non-GLP) and on the results of the first and second experiments which had to be repeated for technical reasons.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 0.1 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

For details see executive summary

Results with reference substance (positive control):

NA

For details see executive summary

Validity criteria fulfilled:

yes

Conclusions:

The test item had no toxic effects on survival and reproduction of Daphnia magna after the exposure period of 21 days up to the solubility limit of the test item in the test water. Thus, the 21-day NOEC of the test item was determined to be at least 100 mg/L nominal (mean measured: 0.10 μg/L). The 21-day LOEC and EC50 for the inhibition of the reproduction rate of the daphnids were above the water solubility limit of the test item.

Executive summary:

Purpose

The purpose of this study was to evaluate toxic effects of the test item on survival and reproduction of Daphnia magna during an exposure period of three weeks.

Study design

For this purpose, a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (1998): “Daphnia magna Reproduction Test” and the EU Commission Directive 92/69/EEC, C.20: “Daphnia magna Reproduction Test was performed. A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to the solubility limit of the test item in test water.
Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was equilibrated for 24 hours in a separating funnel in the dark and the layers with dissolved and undissolved test item were separated. The part of the dispersion with the maximum of dissolved test item was filtered. A single loading rate of the test item of 100 mg/L was tested.
Additionally, a control was tested in parallel (test water without addition of the test item).
The test method is based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

 

Result

The measured test item concentrations in the freshly prepared test medium at the start of the test medium renewal periods were between 0.025 and 0.43 μg/L. In the stability control samples (48 and 72 hours), the measured concentrations were between < LOQ and 0.13 μg/L (with and without food particles and daphnids). The low recoveries at the end of the test medium renewal periods were considered to be caused by the degradation of the test item in the test water as generally expected for antioxidants.

The biological results were based on the loading rate and the mean measured concentration of 0.10 μg/L calculated as a time-weighted mean of the test item concentration measured at the start of the test medium renewals and the concentrations measured in the stability control samples without food at the end of the renewal periods.

The test item had no toxic effects on survival and reproduction of Daphnia magna after the exposure period of 21 days up to the solubility limit of the test item in test water. Thus, the 21-day NOEC of the test item was determined to be at least 100 mg/L (mean measured: 0.10 μg/L). The 21-day LOEC and EC50 for the inhibition of the reproduction rate of the daphnids were above the water solubility limit of the test item.

Summary of the results after 21 days of exposure of the test animals:

	Treatment
	Control	Loading rate 100 mg/L
Mortality (%) after 21 days of exposure	5	5
Mean reproduction rate (living offspring per surviving adult)	124.9	132.2
Mean reproduction rate in % of control	100	106

Conclusion

The test item had no toxic effects on survival and reproduction of Daphnia magna after the exposure period of 21 days up to the solubility limit of the test item in the test water. Thus, the 21-day NOEC of the test item was determined to be at least 100 mg/L (mean measured: 0.10 μg/L). The 21-day LOEC was above the water solubility limit of the test item. The 21-day EC50 for the inhibition of the reproduction rate of the daphnids was above the water solubility limit of the test item.

Description of key information

The test item had no toxic effects on survival and reproduction of Daphnia magna after the exposure period of 21 days up to the solubility limit of the test item in the test water. Thus, the 21-day NOEC of the test item was determined to be at least 100 mg/L (mean measured: 0.10 μg/L). The 21-day LOEC was above the water solubility limit of the test item. The 21-day EC50 for the inhibition of the reproduction rate of the daphnids was above the water solubility limit of the test item.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 100 mg/L

Additional information

Purpose

The purpose of this study was to evaluate toxic effects of the test item on survival and reproduction of Daphnia magna during an exposure period of three weeks.

Study design

For this purpose, a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (1998): “Daphnia magna Reproduction Test” and the EU Commission Directive 92/69/EEC, C.20: “Daphnia magna Reproduction Test was performed. A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to the solubility limit of the test item in test water.
Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was equilibrated for 24 hours in a separating funnel in the dark and the layers with dissolved and undissolved test item were separated. The part of the dispersion with the maximum of dissolved test item was filtered. A single loading rate of the test item of 100 mg/L was tested.
Additionally, a control was tested in parallel (test water without addition of the test item).
The test method is based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

 

Result

The measured test item concentrations in the freshly prepared test medium at the start of the test medium renewal periods were between 0.025 and 0.43 μg/L. In the stability control samples (48 and 72 hours), the measured concentrations were between < LOQ and 0.13 μg/L (with and without food particles and daphnids). The low recoveries at the end of the test medium renewal periods were considered to be caused by the degradation of the test item in the test water as generally expected for antioxidants.

The biological results were based on the loading rate and the mean measured concentration of 0.10 μg/L calculated as a time-weighted mean of the test item concentration measured at the start of the test medium renewals and the concentrations measured in the stability control samples without food at the end of the renewal periods.

The test item had no toxic effects on survival and reproduction of Daphnia magna after the exposure period of 21 days up to the solubility limit of the test item in test water. Thus, the 21-day NOEC of the test item was determined to be at least 100 mg/L (mean measured: 0.10 μg/L). The 21-day LOEC and EC50 for the inhibition of the reproduction rate of the daphnids were above the water solubility limit of the test item.

Summary of the results after 21 days of exposure of the test animals:

	Treatment
	Control	Loading rate 100 mg/L
Mortality (%) after 21 days of exposure	5	5
Mean reproduction rate (living offspring per surviving adult)	124.9	132.2
Mean reproduction rate in % of control	100	106

Conclusion

The test item had no toxic effects on survival and reproduction of Daphnia magna after the exposure period of 21 days up to the solubility limit of the test item in the test water. Thus, the 21-day NOEC of the test item was determined to be at least 100 mg/L (mean measured: 0.10 μg/L). The 21-day LOEC was above the water solubility limit of the test item. The 21-day EC50 for the inhibition of the reproduction rate of the daphnids was above the water solubility limit of the test item.