Fatty acids, C6-12, mixed tetraesters with heptanoic acid, pentaerythritol, 3,5,5-trimethylhexanoic acid and valeric acid

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:

key study

Study period:

01 December 2015 - 07 July 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: See remarks

Remarks:

Guideline study without analysis

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

Specific details on test material used for the study:

Trade name: CALESTER CW
Appearance: Colourless to light yellow viscous liquid
Batch: 3220W
Purity/Composition: UVCB
Test substance storage: At room temperature protected from light
Stable under storage conditions: Unknown
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC): Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids
EC Number EC 451-190-0

Analytical monitoring:

no

Remarks:

Due to the low water solubilty of the test substance and ast it absorbs to the walls of containers it was not possible to develop a method of analysis to measure the concentrations of the test substance in the saturated solutions used in the study.

Vehicle:

no

Details on test solutions:

The test substance was a colourless to light yellow viscous liquid and a UVCB substance. The test substance was not completely soluble in M7 test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test substance.

Test solutions were prepared by applying a two-day period of gentle magnetic stirring. The stirring speed was sufficient to cause entire water column to swirl but gentle enough to prevent the formation of an emulsion. Subsequently, the obtained mixtures were allowed to settle overnight. Thereafter the aqueous Water Accommodated Fractions (WAFs) were siphoned off through glass wool and used as the test solutions. The final test solutions were all clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SYSTEM:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclic parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with known history

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia (resting eggs that develop under carapace in response to stress) or discoloured animals and there was no delay in the production of the first brood.

Characteristics: To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.

BREEDING:

Start of each batch: With new-born daphnids, i.e. less than 3 days old, by placing them individually in 50 mL M7- medium

Maximum age of the cultures: 4 weeks

Monitoring of the individual cultures: Three times a week, the young are counted and the parental daphnids are transferred to new media.

Temperature of medium: 18-22°C

Feeding: Daily, a suspension of fresh water algae (Chlorella pyrenoidosa)

Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.

Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

The study duration was 21 days and the test solutions were renewed three times a week

Hardness:

> 140 mg/L (as CaCO3)

Test temperature:

18-22°C, constant within 2°C

pH:

Between 6.0 to 9.0, constant within 1.5 units

Dissolved oxygen:

> 3 mg/L

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

For the definitive main study WAFs were prepared at 0.20 and 1.0 mg/L nominal loading rates.

Details on test conditions:

1 FIRST RANGE-FINDING TEST:
In the first range-finding test the test solutions were prepared by spiking test medium with acetone stock solutions. The spike solutions were prepared by mixing 0.010, 0.10 and 1.0 mL of test item with 1.27 mL of acetone resulting in 0.010 (A), 0.10 (B) and 1.0 (C) gram test item per gram acetone. Subsequently, volumes of 50 mL M7 medium were spiked with 6.4 μL stock A, 6.4 μL stock B and 11.4 μL of stock C to produce treatment levels of 1.0, 10 and 100 mg/L2, respectively. In addition, 6.4 μL acetone was spiked on 50 mL medium (in duplicate) to serve as solvent-control. An untreated control was also included. Thereafter, five daphnids were introduced per vessel.

1.1 Results:
All daphnids introduced to vessels containing test item were instantly trapped at the surface of test solutions. This did not occur in the blank and solvent control. Therefore, this test was terminated and the spike method was considered not suitable for testing. Subsequently a feasibility test was performed with Water Accommodated Fraction (WAF) procedure in order to determine whether daphnids would be also trapped at the surface of WAFs.

2 FEASIBILITY TEST:
The proposed test was meant to determine whether the reproduction test with the test substance was technically feasible. Water Accommodated Fractions (WAFs) were prepared at a loading rate of 1.0 mg/L (in duplicate) applying a 2-day period of magnetic stirring. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous WAFs were collected via tap (discarding an initial amount of the solution to flush the tap, no glass wool was used). WAFs were distributed to test vessels (2 vessels per replicate WAF) and subsequently, 5 daphnids were introduced to each vessel. The behaviour of daphnids was observed shortly after introduction (26 minutes) and after 24 hours.

2.1 Results:
No daphnids were observed to be trapped at the surface of test solutions in this test shortly after introduction and after 24 hours of incubation. Therefore, this method was considered suitable for the definitive Reproduction test,

3 SECOND RANGE-FINDING TEST:
The second range-finding test was performed with the WAF method. The loading rates of the test substance tested were: 0.20, 0.50 and 1.0 mg/L. Test solutions were prepared similar as in the definitive reproduction test. A control group was also included. Each loading rate consisted of two replicates containing a total of ten daphnids (five each). The total test period was ten days. Test conditions were held as similar as possible to those applied in the reproduction test. Test solutions were renewed on days 1, 3 and 6 during the test. The provided food did not contain Marinure (Marinure, Glenside Group, United Kingdom).

3.1 Results:
Following the difficulties observed in the first range finder test, which caused the daphnids to be trapped in the meniscus of solutions made with acetone as a carrier solvent, evidenced an issue related to a modified surface tension. As a result (and following a feasibility test), a second range finding test was conducted using WAFs. There was no mortality of adult daphnids and no delay in the onset of reproduction during the 10-day range finding test. Test conditions during the range-finding test were maintained within the limits prescribed by the study plan.

4 DEFINITIVE REPRODUCTION TEST:
4.1 Treatments
Test substance: WAFs prepared at 0.20 and 1.0 mg/L
Controls: Test medium without test item or other additives

4.2 Test procedure and conditions:
Test duration: 21 days
Test type: Semi-static
Frequency of renewal: Three times a week
Test vessels Volume: 60 mL (6 x Ø 3.5 cm), all-glass covered with a Perspex plate. Vessels were pre-incubated with respective loading rates and reused during the test in order to minimise adsorption to glass.
Medium: M74
Experimental design: At the start of the experiment (nominal day 0), 10 neonate daphnids, less than one day old, per group were divided over ten vessels each containing a minimum of 50 mL test medium. The control group consisted of 20 daphnids.
Light: 16 h photoperiod daily; intensity at the start: 523-699 lux, intensity at the end: 707-737 lux
Feeding: Twice daily an amount of 0.30 mg C/daphnia/day in a form of a mixture of Chlorella pyrenoidosa suspension combined with Marinure (TOC ratio of this mixture was 5:1). On weekend days, an amount of 0.60 mg C/daphnia/day was added in one single feed.
Temperature: 18-22°C, constant within 2°C
Oxygen concentration: > 3 mg/L
pH: Between 6.0 to 9.0, constant within 1.5 units.
Hardness (blank-medium): > 140 mg/L (as CaCO3).

4.3 Sampling for analysis of test concentratoins:
No sampling for analysis of the test substance in test media was included in this study. The analysis was technically not possible.

4.4 Measuring and recordings:
Parental daphnids:-
Condition: Every workday the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
Presence of eggs in the brood pouch: Every workday
Body length: At the end of the test.

Offspring:-
Appearance of first brood: When observed.
New-born daphnids: Every workday the number of neonates was counted and their condition recorded. Thereafter the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.

Test medium:-
Temperature, oxygen and pH: At the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids.
Hardness: Once a week in fresh and old media from the control and the highest test concentration
Light: At the start and the end of the test

5 Interpretation
5.1 Data handling
Statistical analysis:-
The following statistical procedures were used to determine the NOEL for reproduction and growth:
a) Data distribution: Shapiro-Wilk´s Test
b) Homogeneity of variance: Levene´s Test (with Residuals)
Differences between treatments and the control: Dunnett`s Multiple t-test Procedure (oneside smaller) after trend analysis by contrasts (Monotonicity of Treatment level/Response). No statistical analysis was performed for mortality of parental daphnids as the mortality in the highest group did not exceed 10%, which is considered biologically not relevant and as part of normal variability. The guideline allows 20% mortality in the control treatment.

EL-values for reproduction:-
No EL10 and EL50 values could not be calculated as the observed effects were lower than 10%.All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

5.2 Acceptability of the test
1. The mortality of the parent animals (female Daphnia) in the control did not exceed 20% at the end of the test (0%).
2. The average cumulative number of young per female in the controls after 21 days was ≥ 60 (199 ± 18% CV).

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

Conditions on parental daphnids:
None of the daphnids exposed to the control treatment and WAF prepared at 0.20 mg/L died during the exposure. In the highest loading rate, one daphnid was found dead on day 9 of exposure. It should be noted that the guideline allows 20% mortality in the control treatment, thus the observed effect is considered biologically insignificant.

Time to first reproduction:
There was no delay in the onset of reproduction at any of the groups tested.

Reproduction:
On average, 199 offspring were produced per surviving daphnid in the control treatment. The reproduction in the WAFs was slightly higher. There was an insignificant number of immobile offspring and aborted eggs observed in this study. No ephippia were observed in this study.

Body length:
The growth of daphnids was not affected by the presence of the test substance.

Experimental condistions:
The pH remained within the range of 7.6 to 8.3 throughout the test and thus was maintained within the limits prescribed by the study plan (6.0-9.0, constant within 1.5 units).

The oxygen concentration in all test solutions remained within the range of 5.8 to 10 mg/L during the exposure period and thus complied with the requirements as laid down in the study plan (> 3 mg/L).

The temperatures in the test media varied between 20 and 21°C. The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Total hardness varied between 196 and 250 mg calcium carbonate per litre, and thus complied to the requirements as laid down in the study plan (>140 mg CaCO3 per liter).

No dissolved organic carbon could be detected in M7 medium. This complied with therequirements as set out in the guidelines (TOC < 2 mg/L).

Reported statistics and error estimates:

Shapiro-Wilk´s Test on Normal Distribution:
Results:
Number of residuals = 37
Shapiro-Wilk´s W = 0.940
p(W) = 0.046
p(W) is greater than the selected significance level of 0.010; thus treatment data do not significantly deviate from normal distribution.
Normality check passed (p > 0.01).

Levene´s Test on Variance Homogeneity (with Residuals):
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check passed (p > 0.01).
Normal-distribution and variance-homogeneity requirements are fulfilled.
A parametric multiple test is advisable.
The analysis of contrasts did not reveal a linear trend, thus the selected Williams test was replaced by Dunnett test.

Dunnett`s Multiple t-test Procedure:
The NOEL appears to be higher than or equal 1 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion: the test substance did not affect reproduction or growth of Daphnia magna at a loading rate of 1.0 mg/L after 21 days of exposure (NOELR, No Observed Effect Loading rate).The 21-day EL10 and EL50 for both, reproduction and growth, were beyond the range tested, i.e. exceeded loading rate of 1.0 mg/L.

Executive summary:

Daphnia magna, 21-day reproduction study (semi-static, without analysis).

The study procedures were based on the OECD guidelines for Testing of Chemicals: Guideline No. 211, 2012. In addition, the procedures were designed to meet the test methods and validity criteria of the Commission Regulation (EC) No 440/2008 Part C.20, 2008 and the OECD guidance document number 23, 2000.

The batch of test item tested was a colourless to light yellow viscous liquid and a UVCB (unknown variable composition with biological activity) substance. The test item was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test substance.

Water Accommodated Fractions (WAFs) were prepared at loading rates of 0.20 and 1.0 mg/L and used as test concentrations, from now on referred to as treatment levels or loading rates. The final test solutions were all clear and colourless.

The reproduction test was performed in a semi-static system, included 10 vessels per test loading rate and 20 vessels for an untreated control group. Each of the vessels contained one neonate (<24h old) Daphnia magna in 50 mL test medium. The study duration was 21 days and the test solutions were renewed three times a week. The daphnids were fed on a daily basis with a Chlorella pyrenoidosa and Marinure mixture. The condition of the parental daphnids was recorded every workday, and during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was also recorded. At the end of the test, the lengths of the surviving parental daphnids were measured.

Glassware was reused during the final test in order to minimise adsorption of the test substance. The study met the acceptability criteria prescribed by the study plan and was considered valid. No significant mortality (>10%) was observed in any of the groups tested during the exposure. The average cumulative number of young per female in the control after 21 days was 199 ± 18 (%CV). The reproduction in test groups was not different than in the control treatment. Growth of daphnids exposed to the test substance was similar to those exposed to the control treatment.

Effect parameters (mg/L) obtained in this study are summarized in the table below;

Parameter	Loading Rate (mg/L)
NOELR for reproduction	1.0
EL10 for reproduction	>1.0
EL50 for reproduction	>1.0
NOELR for mortality	1.0
NOELR for growth	1.0

In conclusion the test substance did not affect reproduction or growth of Daphnia magna at a loading rate of 1.0 mg/L after 21 days of exposure (NOELR, No Observed Effect Loading rate).

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

1 substance available for read across

Adequacy of study:

weight of evidence

Justification for type of information:

see the attached justification in section 13 for the full details.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Validity criteria fulfilled:

not applicable

Remarks:

read across

Conclusions:

The read across for substance, CAS: 156558-98-4; EC: 451-190-0; is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of long-term toxicity to invertebrates. The EL50/NOEC for the substance based on the mean of the information available is deemed to be > 1mg/L.

Description of key information

The read across for substance, CAS: 156558-98-4; EC: 451-190-0; is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of long-term toxicity to invertebrates. The EL50/NOEC for the substance based on the mean of the information available is deemed to be > 1mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 mg/L

Additional information

In a OECD 211 Daphnia magna reproduction test the test substance Pentaerythritol tetraesters of n-C5, n-C7, n-C8, i-C9 and n-C10 fatty acids did not affect reproduction or growth of Daphnia magna at a loading rate of 1.0 mg/L after 21 days of exposure (NOELR, No Observed Effect Loading rate).The 21-day EL10 and EL50 for both, reproduction and growth, were beyond the range tested, i.e. exceeded loading rate of 1.0 mg/L.