Glycerides, C16-18 and C18-unsatd. mono- and di-, citrates

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:

18 Feb - 10 Mar 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)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control, 10, 20, 30, 60, 100 mg/L loading rates
- Sampling method: Samples were collected in 100 mL glass bottles from each test concentration and the control prepared in additional test beakers without daphnids and algae. They were collected at the initiation of the test (0 h), at each renewal (old and new test solutions) and at the termination of the test. Duplicate samples of new test solutions, approx. 2 x 100 mL, were collected and 100 mL sample from the old test solutions. A small amount of hydrochloric acid was added to a pH <2.
- Sample storage conditions before analysis: The samples were stored at 4 °C ± 2 °C until the end of the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Water Accommodated fractions of the test item were prepared according to OECD 23. For each loading rate, a WAF was prepared individually. The series of WAFs were prepared in 2 L glass bottles by weighing out the required amounts of the test item on a cover glass, adding it to 2000 mL test medium and gentle stirring for 22 ± 2 h. After stirring, the mixtures were allowed to settle for 22 ± 2 h, due to the appearance of small particles in the water column. 1100 mL of the mid fractions was transferred to a series of new glass beakers by syphoning, i.e. by use of a silicone tube connected to a glass tube. pH was measured to be 7.6-7.9 in the mid-fractions and used directly as test solutions. Due to the presence of precipitations in the bottles the hardness of the test solutions was measured. The hardness was within the required range.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium control

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: in-house culture since 1979
- Method of breeding: The culture is fed with Pseudokirchneriella subcapitata (clone: NIVA, CHL 1) three times a day by a peristaltic pump system. The culture is renewed once a week by discharging the oldest culture and preparing a new culture with one to four day old offspring taken from a three week old culture.
Feeding during test
- Food type: Pseudokirchneriella subcapitata
- Amount: approx. 0.15 mg C/adult animal/day until day 8; 0.2 mg C/adult animal/day after day 8
- Frequency: at each media renewal

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

261 - 262 mg CaCO3/L

Test temperature:

20.2 ± 0.1 °C

pH:

7.7 - 8.6

Dissolved oxygen:

92 - 100% O2 saturation

Nominal and measured concentrations:

nominal: control, 10, 20, 30, 60, 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers covered with Parafilm filled with 60 mL of test medium
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): medium was changed (e.g. female was transferred to a new vessesl with fresh medium) every monday, wednesday and friday
- 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: according to guideline
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
- Light intensity: 11.9 ± 1.3 µmol x m-2 x s-1

EFFECT PARAMETERS MEASURED
- Number of offspring alive
- Mortality among parent animals
- Time at which the first offspring were present
- Aborted broods and dead offspring
All parameters were recorded at each renewal of test solutions.

RANGE-FINDING STUDY
- A range-finding test was performed (non-GLP) in order to define the loading rates for the test.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: time to first brood

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

other: time to first brood

Details on results:

- Mortality of parent animals: 10% in control, 10 mg/L and 20 mg/L treatments
- Number of males and females (parental): only females
- Time to first brood release or time to hatch: 8.9 d (control), 8.5 d (10 mg/L), 10 d (20 mg/L), 9.9 d (30 mg/L), 9.5 d (60 mg/L), 14.6 d (100 mg/L)
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

The statistical analyses were performed using the free software R (version 3.0.3 - 2014-03-06). The NOELR and LOELR values were determined by use of the Dunnett’s test. The LOELR value (p>0.05) was determined as the highest tested concentration, at which a significant effect was observed when compared to the control.

Table 1: Summary of endpoints observed in the reproduction test with Daphnia magna.

Nominal Loading rate (mg/L WAF)	Average number of offspring per live female	Mortality among parent animals (%)	Average time to which the first offspring were present (days)
Control	92.0	10	8.9
10	118	10	8.5
20	83.3	10	10.0
30	44.7	0	9.9
60	71.2	0	9.5
100	9.7	0	14.6

Table 2: Results from the NVOC analysis of the test solutions in mg C/L. Results are corrected by subtraction of the control values as background carbon concentration.

Day	0	1	1	4	8	11	11	13	18	20	20	21
WAF [mg/L]	New	Old	New	Old	New	Old	New	Old	New	Old	New	Old
Control	0	0	-	-	0	0	-	-	-	-	0	0
10	0.10	0.10	-	-	0.05	0.05	-	-	-	-	0.22	0.18
20	0.04	0.01	0.01	0.03	0.10	0.08	0.03	0.07	0.09	0.11	0.36	0.46
30	0.09	0.14	0.36	0.30	0.31	0.21	0.51	0.50	0.74	0.74	0.66	0.66
60	0.25	0.28	0.17	0.14	0.47	0.54	0.64	0.54	0.64	0.64	0.66	0.56
100	0.84	0.94	1.04	1.14	0.21	0.19	1.34	1.34	1.04	1.04	1.36	1.36

The results show that when preparing WAFs of the test item with low solubility some variation is observed between new solutions. Furthermore, the results show that the soluble part of the test item is stable between the renewals (new and old).

Validity criteria fulfilled:

yes

Description of key information

One experimental study is available investigating the long-term effects of Glycerides, C16-18 and C18-unsatd. mono- and di-, citrates (CAS 91052-16-3) to aquatic invertebrates (Kamper, 2016). The study was performed according to OECD 211 (GLP) under semi-static conditions with the water flea Daphnia magna. Water accommodated fractions of 10, 20, 30, 60 and 100 mg/L were prepared individually in accordance to OECD Guidance 23. At each renewal period of the test medium the number of offspring, mortality of parent animals and time of first brood were recorded. After 21 d an inhibition of reproduction was recorded at the highest loading rate of 100 mg/L resulting in a NOELR (21 d) of 60 mg/L. However, the loading rate of 100 mg/L is far above water solubility which was calculated to be 1.86E-37 to 1.2721 mg/L based on QSAR calculations. All other loading rates did not cause any significant effects on the number of offspring or time to first brood. Thus, this result may overestimate the possible effects to aquatic invertebrates. It is possible that after transferring the mid fractions of the settled test solution to the exposure vessels undissolved particles were transferred as well which might have caused physical effects. Under environmental conditions this effect is not likely to occur.