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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 January 2013 to 12 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted in accordance with OECD and EU test guidelines and was conducted by an accredited GLP laboratory
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
For determination of the test item concentrations, duplicate samples were taken from the freshly prepared test media of all test concentrations and from the control at one treatment period of the first, second and last week of the test (Day 0, 7 and 16, respectively).
Vehicle:
yes
Details on test solutions:
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 at Harlan Laboratories in culture medium identical to the medium used for the test. The temperature and light conditions were identical to those of the test. During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany). Each stock animal was maintained separately in a 100-mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. 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 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 at Harlan Laboratories in culture medium identical to the medium used for the test. The temperature and light conditions were identical to those of the test. During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany). Each stock animal was maintained separately in a 100-mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. 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:
other: Reconstituted water (Elendt M7 medium)
Remarks:
see "Any other information on materials and methods incl. tables" for more details.
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
Not applicable
Hardness:
2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
Maintained between 20-21°C
pH:
Between 7.6 and 8.0
Dissolved oxygen:
At leaste .7 mg/L
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
The following nominal concentrations of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) were tested: the undiluted filtrate with the loading rate of 100 mg/L and dilutions of 1:3.2, 1:10, 1:32 and 1:100. Additionally, a control was tested in parallel (test water without test item).
Details on test 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 20-21 °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 390 and 560 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 each working day (Monday through Friday) with a food mixture containing a suspension of green algae of the species Scenedesmus subspicatus (freshly grown at Harlan Laboratories) and a fish food suspension. The fish food suspension was fed in addition to the algal food, because a toxic effect of the test item on the algae could not be excluded and to reduce the amount of algal food to prevent an increase of the pH values in the test media due to algal growth. The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, 49304 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.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.013 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.013 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
reproduction
Details on results:
Taking into account the effects on survival and reproduction of the test animals, the highest concentration of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) tested without toxic effects after the exposure period of 21 days (21-day NOEC) was the dilution 1:3.2 (mean measured concentration of 0.0043 mg/L).The lowest concentration tested with toxic effects (21-day LOEC) was determined to be the undiluted filtrate (highest mean measured concentration of 0.013 mg/L) due to the significantly reduced mean reproduction rate of Daphnia magna at this test concentration.The 21-day EC50 for the inhibition of the reproduction rate of the daphnids was higher than 0.013 mg/L.
Results with reference substance (positive control):
Positive control not used.
Reported statistics and error estimates:
In the control and up to and including the highest test concentration, the survival of the test animals at the end of the test was at least 90% or higher. Mortality up to 20% is regarded as natural and tolerated by the test guideline. Thus, the survival of Daphnia magna over 21 days was not affected by the test item up to and including the highest mean measured concentration of 0.013 mg/L. The first young offspring released from their parent animals were recorded in the control and at all test concentrations at observation on Day 9. Thus, the time of the first brood was not affected by the test item up to and including the highest mean measured concentration of 0.013 mg/L. The mean reproduction rate of the daphnids in the control was 163 ± 6.7 living offspring per surviving adult (mean ± standard deviation). No significant inhibitory effect of the test item on the mean reproduction rate was determined up to and including the dilution 1:3.2 (mean measured concentration of 0.0043 mg/L). First at the highest mean measured concentration of 0.013 mg/L, the mean reproduction rate of surviving daphnids was statistically significantly reduced to in the mean 115 living offspring (compared to the control, Dunnett t-test, one-sided smaller, α = 0.05). With the exception of the reduced reproduction rates, no visible abnormalities were observed at the test animals during the test.

General Results

During the test period, the pH of the test media and control ranged from 7.6 to 8.0. The dissolved oxygen concentrations were at least 7.7 mg/L. The water temperature was between 20 and 21 °C during the test period.

 

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test medium renewal periods.

Analytical Results

The concentrations in the freshly prepared test media of the relevant concentrations (NOEC and LOEC) were 0.0049-0.0052 mg/L (dilution 1:3.2) and 0.012-0.017 mg/L (undiluted filtrate), respectively.

 

In the aged test media samples without food, the measured concentrations decreased and were 0.0027-0.0043 mg/L (dilution 1:3.2) and 0.0090-0.015 mg/L (undiluted filtrate), respectively or 52 to 84% of the initially measured values.

 

Under the conditions of the test, 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) was not stable during the test medium renewal periods of two and three days. Therefore, all reported biological results are related to the mean measured concentrations of the test item.

 

In the aged test media samples with food, the measured concentrations were lower than in the stability control samples without food particles. Thus, a part of the test item had obviously adsorbed onto the food particles. However, since filter feeding test animals asDaphniamay take up the test item also from ingested food, the decrease of the test item concentrations in the test media due to adsorption onto food is not taken into account.

 

Therefore the mean measured test item concentrations during the test period were calculated as the time-weighted means over the measurements per test concentration at the start and the end of the renewal periods. For the end of the renewal periods, the samples without food were taken into account.

 

The biological results are based on mean measured test item concentrations (calculated as the time-weighted means over all measurements per test concentration without food):

Dilution

Mean measured test item concentration (time-weighted means)

(mg/L)

1:100

---

1:32

---

1:10

---

1:3.2

0.0043

Undiluted filtrate (loading rate 100 mg/L)

0.013

 

Validation of Analytical Method Specificity

The biological control samples and an analyzed analytical blank (test water) did not significantly affect the chromatogram at the retention time of the test item. The calibration solutions contained a peak specific for the test item, whose area changed accordingly with known concentration.

Calibration

The R² fits of the calibration curves used were 1.0000 and 0.9999. This reflects the linearity of the analytical system within the calibration range of 0.0273 to 6.55 mg test item/L.

Accuracy (Recovery) and Precision

Concurrent with the sample analysis, a set of recovery samples accurately fortified at relevant concentrations of test item (0.00204 and 0.0164 mg test item/L), was prepared five-fold and analyzed.. The chromatograms of the recovery samples were evaluated after subtracting the mean value of the counts of the analytical blanks. The samples were directly evaluated, as the disturbance of the baseline was not relevant here. The average recoveries were found to be 87% and 100% of the spiked values with relative standard deviations of 15% and 1%, respectively. The method was considered to be sufficiently accurate and precise for the purposes of this test. The test sample results were not corrected for recovery.

Limit of Quantification

The limit of quantification for the test item in the test samples was derived from the lowest recovery sample. The LOQ is 0.002 mg test item/L.

Validity criteria fulfilled:
yes
Conclusions:
Taking into account the effects on survival and reproduction of the test animals, the highest concentration of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) tested without toxic effects after the exposure period of 21 days (21-day NOEC) was the dilution 1:3.2 (mean measured concentration of 0.0043 mg/L).The lowest concentration tested with toxic effects (21-day LOEC) was determined to be the undiluted filtrate (highest mean measured concentration of 0.013 mg/L) due to the significantly reduced mean reproduction rate of Daphnia magna at this test concentration.The 21-day EC50 for the inhibition of the reproduction rate of the daphnids was higher than 0.013 mg/L.
Executive summary:

The effect of the test item 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) on the survival and reproduction of Daphnia magna was investigated in a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (2008): “Daphnia magna Reproduction Test”, the EU Commission Directive 92/69/EEC, C.20: “Daphnia magna Reproduction Test” and the Commission Regulation (EC) No 440/2008, C.20: “Daphnia magna Reproduction Test”.

 

Due to the low water solubility of the test item, a supersaturated dispersion of the test item with a loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours.

 

Then, the dispersion was filtered. The undiluted filtrate with the loading rate of 100 mg/L and dilutions of 1:3.2, 1:10, 1:32 and 1:100 of the undiluted filtrate with the loading rate of 100 mg/L were tested. Additionally, a control was tested in parallel.

 

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

 

The concentrations in the freshly prepared test media of the relevant concentrations (NOEC and LOEC) were 0.0049-0.0052 mg/L (dilution 1:3.2) and 0.012-0.017 mg/L (undiluted filtrate), respectively.

 

In the aged test media samples without food, the measured concentrations decreased and were 0.0027-0.0043 mg/L (dilution 1:3.2) and 0.0090-0.015 mg/L (undiluted filtrate).

Under the conditions of the test, 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) was not stable during the test medium renewal periods of two and three days. Therefore, all reported biological results are related to the mean measured concentrations of the test item.

 

Therefore the mean measured test item concentrations during the test period were calculated as the time-weighted means over the measurements per test concentration at the start and the end of the renewal periods. For the end of the renewal periods, the samples without food were taken into account.

The biological results are based on mean measured test item concentrations (calculated as the time-weighted means over all measurements per test concentration without food):

Dilution

Mean measured test item concentration (time-weighted means, mg/L)

1:100

not analyzed

1:32

not analyzed

1:10

not analyzed

1:3.2

0.0043

Undiluted filtrate (loading rate 100 mg/L)

0.013

 

 

 

 

Control

Treatment / Dilution

(in brackets mean measured concentration, mg/L)

 

1:100

(n.a.)

 

1:32

(n.a.)

 

1:10

(n.a.)

 

1:3.2

(0.0043)

Undiluted filtrate (0.013)

Mortality (%) after 21 days of exposure

0

0

0

0

10

0

Mean reproduction rate (living offspring per surviving adult)

 

163.0

 

151.9

 

148.3

 

150.3

 

148.0

 

115.0*

Mean reproduction rate in % of control

100.0

93.2

91.0

92.2

90.8

70.6

* significantly lower than the control value

n.a. not analysed

 

In conclusion, taking into account the effects of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) on survival and reproduction of the test animals, the 21-day NOEC was the dilution 1:3.2 (mean measured concentration of 0.0043 mg/L). The 21-day LOEC was the undiluted filtrate (mean measured concentration of 0.013 mg/L), due to the statistically significantly reduced mean reproduction rate of Daphnia magna at this test concentration.

The 21-day EC50 for the inhibition of the reproduction rate of the daphnids was higher than 0.013 mg/L. The substance is considered to not immobilise Daphnia magna at the limit of solubility in the test media and no toxicity effects have been noted at the 21 -day LOEC or the 21 -day NOEC.

 

Description of key information

Long-term toxicity to aquatic invertebrates

Key values were determined in a GLP accredited laboratory study “Daphnia magna Reproduction test” using the following test guidelines: OECD Guidelines for the Testing of Chemicals, No. 211, October 03, 2008 and EU Commission Regulation (EC) No 440/2008, C.20. The key values for 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) were calculated as:

21 d NOEC (reproduction) 0.0043 mg/L (mean, dilution 1:3.2)

21 d NOEC (survival) 0.0043 mg/L (mean, dilution 1:3.2)

21 d LOEC (reproduction) 0.013 mg/L (mean, undiluted)

21 d EC50 (reproduction) > 0.013 mg/L (mean, undiluted)

The substance is considered to not immobilise Daphnia magna at the limit of solubility in the test media and no toxicity effects have been noted at the 21 -day LOEC or the 21 -day NOEC.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.004 mg/L

Additional information