Isodecyl pivalate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 Apr - 16 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Due to the difficult dissolving properties, a water accommodated fraction (WAF) of the test item was used. The WAF was prepared in accordance with the OECD guidance No. 23. A concentrated stock solution of the test item was prepared by dissolving 4 mg of the test item in 2 L of holding and dilution water. The solution was stirred at 100 rpm for 24 hours at room temperature (21 ± 2 °C). The WAF solution was then be left to settle for 24 hours. The saturated aqueous phase was drained from the bottom of the bottle via a drain port, and the first 100 mL discarded. The remaining eluate formed the test loading of 2 mg/L, and was also drained via the drain port. The upper part of the bottle content, potentially containing the non-dissolved part of the substance, was not drained.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: daphnid
- Source: test site breeding
- Age of parental stock: between 4 and 24 h old
- Feeding during test: feeding occurred daily after the removal of juveniles and at exchange of test solutions. The concentration of carbon (mg carbon/L) in the algal culture was measured by optical density at 585 nm and compared to a calibrated nomograph to calculate the volume required for feeding of adult daphnids.
- Food type: fed concentrated suspensions of living cells of the unicellular alga Desmodesmus subspicatus.ACCLIMATION- Acclimation period: to maintain the stock culture, batches of 30 - 50 adult daphnids were held at room temperature in approximately 1.8 L of dilution water for one week. During this week, the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus containing Artemio Fluid JBL)
- Acclimation conditions: same as test

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: For use in the test, newborn Daphnia (between 4 and 24 h old) were removed by wide-bore pipette (to avoid damage) and isolated in fresh dilution water for at least 1 h prior to being added to the test vessels.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

1.0 mmol/L

Test temperature:

20.0 – 20.7°C with less than 2 °C variation

pH:

7.92 – 9.62 and did not deviate by more than 1.5 units

Dissolved oxygen:

5.3 – 9.7 mg/L

Nominal and measured concentrations:

nominal: control and 2 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: the test vessels were 100 mL glass flasks stoppered with silicone plugs.
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified local tap water
- Total organic carbon: 0.4506 mg/L
- Metals: cadmium <0.329 µg/L, copper <1.73 µg/L
- Chlorine: <0.02 mg/L
- Alkalinity: 1.6 mmol/L
- Ca/mg ratio: 0.7/0.3
- Conductivity: 207.7 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: monthly

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: light/dark cycle of 16/8 hours
- Light intensity: between 784-871 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The number of juvenile D. magna per replicate was recorded daily from day 6 onwards. Mortality, abnormalities in appearance, behaviour, condition (including male sex) or presence of winter eggs (ephippium) were also recorded. At study termination, body length (excluding the anal spine) of the adults in all treatments without significant mortality was captured by digital photography. Measurements of the individual daphnids were done by image analysis software.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: inhibition of reproduction: 29.9% at 100 mg/L

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

other: NOEL

Effect conc.:

< 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(WAF)

Basis for effect:

immobilisation

Details on results:

- Mortality of parent animals: a single adult mortality occurred on before day 6 of the test in the control treatment (10% immobility), most likely due to handling damage.
- No. of offspring produced per female: cumulative 47 +/- 7.9
- Body length and weight of parent animals: 3.76 +/- 2.87 mm
- Type and number of morphological abnormalities: no abnormalities
- Type and number of behavioural abnormalities: no abnormalities
- Time to first brood release or time to hatch: the mean age to first brood was day 9.9 for the control and day 11.4 for the test loading

Reported statistics and error estimates:

The mean coefficient of variation for the control reproduction was 21.8%. The OECD 211 guideline recommends this parameter be ≤ 25%. Reproduction (juvenile production) was inhibited by 27.7% by the test loading of 2.0 mg/L (p=0.004) (29.9% by 100 mg/L in the previous test). A single adult mortality occurred on before day 6 of the test in the control treatment (10% immobility), most likely due to handling damage.

In a preliminary study with a nominal loading rate of 100 mg/L was applied resulting in a sinificant inhibition of reproduction (29.9% inhibition). This study was not finalized and it was repeated with the lower loading rate of nominal 2 mg/L since the observed effects might be due to oversaturation and physical effects.

The cumulative number of offspring per replicate of the control at test end was 65 for the control and 47 for the test loading. The NOEL could not be determined as a significant effect was demonstrated (Table 1).

Table1:   Survival, growth and reproduction endpoint averages forDaphnia magnaat the end of the 21 d exposure period.

Nominal treatment loading	Immobility		Length	Age at first brood	Cumulative offspring per female
(mg/L)	(%)		Mean ± SD (mm)	Mean ± SD (days)	Mean ± SD (n)
Control	10		4.20 ± 2.87	9.9 ± 0.73	65 ± 14.3
2.0	40		3.76 ± 2.87	11.4 ± 1.45	47 ± 7.9
NOEL	≥ 2.0 mg/L	n.d.		n.d.	n.d.

n.d.: not able to be determined from the data as there were effects at the response level.

Within the 2.0 mg/L treatment, the NOEL appears to be higher than or equal to 2.0 mg/L despite 40% immobility (p=0.152) (Table 1).On days 15, 16 and 19 unhatched eggs were observed in the 2.0 mg/L treatment.

 

The mean age to first brood was day 9.9 for the control and day 11.4 for the 2.0 mg/L treatment (p=0.007). The NOEL could not be determined as a significant effect was demonstrated.

 

Growth (body length) was reduced 10.6% by the test loading of 2 mg/L (p=0.023). The NOEL could not be determined as a significant effect was demonstrated (Table 2).

Table 3: Parental lengths at day 21, [mm].

Replicate	Control	2.0 mg/L
1	*	*
2	4.62	3.97
3	3.89	3.21
4	3.85	*
5	3.98	4.19
6	4.05	*
7	4.68	3.95
8	4.78	3.86
9	3.86	3.36
10	4.09	*
# Replicates	9	6
Mean	4.20	3.76
Std.Dev	0.38	0.38

Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates.

 * Adult female did not survive until test end

Validity criteria fulfilled:

yes

Conclusions:

The 21-day-chronic toxicity of isodecyl neopentanoate to Daphnia magna was examined in a semi-static limit test. The NOEC, based on immobility was ≥ 2 mg/L. The NOEL for reproduction, effects on growth or mean age to first brood could not be determined.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 - 28 Sep 2016

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:

02 Oct 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany (31 May 2016)

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

- Concentrations: Control and limit concentration (100 mg/L)
- Sampling method: 250 mL samples were taken from fresh and aged test solutions.
- Sample storage conditions before analysis: Chemical analysis of the processed samples was performed immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item is a UVCB which is insoluble in water. Therefore, the test item was applied as Water Accommodated Fraction (WAF) according to OECD guidance No. 23 with a WAF loading rate of 100 mg test item/L.
- Preparation of Water Accommodated Fraction (WAF): The loading rate of 100 mg test item/L was prepared by pipetting 174.8 µL test item into a WAF-bottle containing 0.5 L water, which was immediately filled up to 1.5 L with water. The WAF was prepared by stirring the water for 48 h at room temperature. On weekends the WAF was stirred for 72 h at room temperature. The water was stirred slowly to avoid bubble and foam formation using a star shape magnetic stirring bar. After stirring, the saturated aqueous phase was taken out of the drain port. The first fraction (200 mL) was discarded. Approx. 800 mL were centrifuged at 9500 g for 25 min. Polypropylene tubes were used for centrifugatin. After this step, they were lancinated with a solid syring - connected with a flexible tube - to purge the lower aqueous layer into a sample vessel. Eluates were checked for irregularities such as foam prior to exposure and filling of the test flasks for toxicity testing. The test solutions were prepared freshly before test start and at each renewal three times a week.
- Controls: Dilution water only
- Evidence of undissolved material: No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age at test start: < 24 h (females)
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test were bred in the laboratory of the testing facility.
- Breeding conditions: Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for 1 week. During this week the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and LiquizellR (HOBBY). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. 30 mL of this suspension was given to 1 L Daphnia medium. The water was changed once per week. Newborn Daphnia were separated by sieving, the first generation was discarded. Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy speciments.
- Feeding during test: The daphnids were fed with suspensions of unicellular alga Desmodesmus subspicatus. The food content in the test suspensions was measured at 585 nm and was kept constant at 0.2 mg C/Daphnia/Day during the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

Control: 1.1 - 1.2 (new medium); 1.2 - 1.3 (aged medium)
Treatment: 1.2 (new medium); 1.2 - 1.3 (aged medium)

Test temperature:

20.5 - 21.6 °C

pH:

Control: 8.13 - 8.84 (aged medium); 8.00 - 8.24 (new medium)
Treatment: 8.09 - 8.87 (aged medium); 8.01 - 8.20 (new medium)

Dissolved oxygen:

Control: 8.23 - 10.62 mg/L (aged medium); 7.45 - 8.50 mg/L (new medium)
Treatment: 8.37 - 10.95 (aged medium): 7.30 - 8.87 mg/L (new medium)

Conductivity:

271 - 272 µS/cm (dilution water)

Nominal and measured concentrations:

100 mg/L (nominal loading rate WAF)
13.34 µg/L (time weighted mean measured initial and mean measured aged concentration at test solution renewal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL round glass beakers filled with 50 mL test solution and covered with glass panes
- Renewal rate of test solution (frequency/flow rate): 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified drinking water was used as holding- and dilution water. The purification included filtration with activated charcoal, passage through a lime-stone column and aeration. To avoid copper contamination, plastic water pipes were used for the testing facilities.
- Conductivity: 271 - 272 µS/cm
- Alcalinity: 2.0 mmol/L
- Total hardness: 1.2 - 1.3 mmol/L
- Ca-hardness: 1.1 mmol
- Mg-hardness: 0.1 - 0.2 mmol/L
- NPOC: 0.33 - 0.40 mg/L
- NO3 = 7 - 8 mg/L
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: Oxygen concentration, pH value and temperature were checked directly before adding the animals and at each water renewal in new and aged test solutions. Hardness was checked 1x a week at water renewal in new and aged control media and test solutions of the highest treatment. Water chemistry data of the dilution water were recorded regularly in the testing facility.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 11.4 - 11.9 µE/(m²*s) or 854 - 895 lux

EFFECT PARAMETERS MEASURED:
- N° of immobile daphnids: Daily
- N° of newborn daphnids per beaker: Daily
- Abnormalities in appearance/condition and behaviour: Daily
- Body length of adults: At study termination (digital photography)

TEST CONCENTRATIONS
- Range finding study: Not specified

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

< 13.34 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Survival of the adults was not affected.

Remarks on result:

other:

Remarks:

The test item has a chronic adverse effect on reproduction of adult Daphnia magna at the tested concentration.

Details on results:

- Cumulative Offspring per Introduced Parent at 21d: Reduced by 19.6 % in the treatment
- Development Rate: : Reduced by 7.9 % in the treatment
- Intrinsic Rate: : Reduced by 6.2 % in the treatment

CONTROL ASSAYS
- Mortality of parent animals: 0 %
- No. of offspring produced per female: 76.5 +/- 8.96
- Body length of parent animals: 4.67 +/- 0.17 mm
- Type and number of morphological abnormalities: None
- Type and number of behavioural abnormalities: None
- Number of males and females (parental): No males
- Age at first brood release: 8.8 +/- 0.48
- Other biological observations: Intrinsic rate of increase 0.302 +/- 0.022 Ind./day

Results with reference substance (positive control):

- Results with reference substance valid? Yes, latest study dates Feb 2016.
- Relevant effect levels: EC50 (24 h) = 1.38 mg/L (95% confidence level: 1.19 - 1.64 mg/L)

Reported statistics and error estimates:

Statistical analyses were performed with ToxRat Pro 3.2.1 (ToxRat Solutions) and included an ANOVA test followed by a Student-t-test, Fisher's Exact Binomial test, William's test or an appropriate non-parametric test for each endpoint. Since only 1 concentration was tested, the concentration-effect relationship and effect concentrations could not be computed and no LOEC, NOEC, EC10 or EC50 could be calculated.

VALIDITY CRITERIA

The test is considered valid (Table 1).

The dissolved oxygen concentration was > 3 mg/L throughout the test. The pH was in the required range of 6 - 9 and did not vary more than by 1.5 units. The coefficient of variation for the mean number of control offspring (11.7%) was ≤ 25%.

Table 1. Validity criteria for OECD 211.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (femaleDaphnia) does not exceed 20% at the end of the test.	Mortality was 0% in the controls.	Yes
The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60 in the control.	The mean n° of offspring was 76.5.	Yes

ANALYTICAL RESULTS

A WAF with a nominal loading rate of 100 mg test item/L was prepared, resulting in a mean measured test item concentration of 34.43 µg/L. During the time interval until renewal of the test solution, the test item concentration decreased below the limit of quantification (7.2 µg/L). Therefore, time-weighted mean concentrations were calculated for the evaluation of the biological parameters and the endpoints. For concentrations < LOQ, a value half of the LOQ (3.6 µg/L) was set.

The time-weighted mean of mean measured initial and mean measured aged concentrations at test renewal was 13.34 µg/L, corresponding to 38.73% of the mean measured initial concentration (Table 2).

Table 2. Concentrations of the test item.

Mean measured initial concentrations [µg/L], mean measured aged concentrations [µg/L], time-weighted mean concentration [µg/L] and percentage of mean measured initial concentration [%]. LOQ = limit of quantification (7.2 µg/L).

Nominal conc.	Treatment
Mean measured initial conc. [μg/L]	34.43
% of mean measured initial conc.	100
Mean measured aged conc. [μg/L]	< LOQ
% of mean measured initial conc.	-
Time weighted mean conc. [μg/L]	13.34
% of mean measured initial conc.	38.73

BIOLOGICAL RESULTS

Results are summarized in Table 3 and Table 4.

Survival / Body Growth / Physical, pathological & Behavioural Symptoms

No significant differences in survival of individuals were observed in the treatment compared to the control. Thus, no mortality or other clinical signs occurred as a result of test item exposure.

Reproduction and population growth

The age of the daphnids was 8.8 d and 9.6 d for control and treatment, respectively. This differences was statistically significant.

The cumulative number of offspring per adult Daphnia ranged from 76.5 in the control to 61.5 in the treatment. This difference in the number of offspring was statistically significant.

The intrinsic rate of increase ranged from 0.302 in the control to 0.283 (mean values) in the treatment. The difference of the intrinsic rate was statistically significant. Thus, they suggest a test item depending effect.

No concentration-response-relationship could be computed since only one concentration was tested. Thus, no NOEC, LOEC or EC values could be calculated.

Table 3. Survival, growth and reproduction data.

SD = standard deviation. TWM = time-weighted mean. n = 10.

Concentration [μg/L]	Parental survival [%]	Growth (length on day 21) (Mean±SD [mm])	Age at first brood (Mean±SD [days])	Cumulative offspring per female (Mean±SD [Ind.])	Intrinsic rate of increase (Mean±SD [Ind./day])
Control	100	4.67 ± 0.17	8.8 ± 0.48	76.5 ± 8.96	0.302 ± 0.022
Treatment 13.34 (TWM)	100	4.69 ± 0.16	9.6 ± 0.88	61.5 ± 12.40	0.283 ± 0.022

 

Table 4. Summary of effects based on the calculated time-weighted mean concentration (TWM).

Concentration [μg/L]	Parental survival	Growth (length on day 21)	Age at first brood	Cumulative offspring per female	Intrinsic rate of increase
Treatment 13.34 (TWM)	not significantly different	not significantly different	significantly different	significantly different	significantly different

 

CONCLUSION

Survival was not affected by exposure to the test item but the reproduction of the survived females was significantly affected at the tested limit concentration of 13.34 µg test item/L (TWM). Thus, the test item showed a chronic adverse effect on the reproduction of adult Daphnia magna under the selected test conditions.

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC(21 d) < 13.34 µg/L (TWM; OECD 211)

Additional information

Two GLP guideline studies according to OECD 211 are available investigating the long-term effects of isodecyl pivalate (CAS 60209-82-7) on Daphnia magna.

The key study (2017) was performed according to OECD guideline 211under semi-static conditions using a water accommodated fraction (WAF) with a nominal loading rate of 100 mg/L. The test solution was renewed three times a week. The test concentrations of fresh and aged test solutions were analytically verfied by Gas Chromatography. The arithmetic mean measured concentrations of fresh test solutions were 34.43μg test item/L. During the time interval until renewal of the test solution, test item concentrations decreased below the limit of quantification (7.2μg/L). The time weighted mean (TWM) of mean measured initial and mean measured aged concentration at test solution renewal was 13.34μg/L, corresponding with 39 % of the arithmetic mean measured concentrations of fresh test solutions. Effects on the survival and growth of Daphnids were not observed. However, a significant effect on the age to first brood, development rate and intrinsic rate was determined. The NOELR (21 d) was set to < 13.34μg/L (measured, TWM) based on the reproduction of D. magna.

The results are supported by a second study (2013) following OECD guideline 211. The test was conducted as a semi-static limit test using a water accommodated fraction (WAF) with a nominal loading rate of 2 mg/L. The test solution was renewed daily. The reproduction was significantly inhibited by 27.7% at the loading rate of 2 mg/L (WAF). Thus, no NOELR (21 d) could be determined but was derived to be < 2 mg/L (WAF, nominal loading rate). However, no effects on mobility were observed throughout the exposure period of 21 days. No turbidity or undissolved test substance was observed in the test vessels. All test solutions were clear throughout the test. Therefore, the effects on reproduction are most likely related to the water soluble part of the substance and not caused by physical effects of undissolved particles.