Fatty acids, C16-18 (even numbered), ammonium salts

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:

10 February 2018 - 07 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

In the range-finding test, a background level of test item was detected in the control samples, QC samples and analytical blanks. It should be noted that tubes, plastic tips, Milli-Q water, solvents and washing liquids used in laboratory all contain fatty acids background level (including stearic acid and palmitic acid). The concentrations detected in test item treated samples were at a comparable level to this background level and no clear dose-related increase of concentrations were observed. Therefore, the results were considered inconclusive. A validated analytical method was used (Test Facility Study No. 519628).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The batch of Fatty acids, C16-18 (even numbered), ammonium salts tested was a white liquid with containing 32.98% active ingredient in water and the item was not soluble in test medium at the loading rates initially prepared. A correction was made for the water content (32.98%) of the test item. A correction factor of 3.0 was used. Concentration/doses are expressed as active ingredient. Water Accommodated Fractions (WAFs) were prepared at a loading rates of 1.0, 10 and 100 mg a.i./L. A 2 - day period of magnetic stirring was applied to accelerate dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Water Accommodated Fractions (WAF) were collected by means of siphoning through glass wool and used as test concentrations. After preparation, volumes of 50 mL were added to each replicate of the respective test group

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, 1820
- Source: In-house laboratory culture with a known history.
- Age of parental stock (mean and range, SD): At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions. Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood. To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old.
- Feeding during test
- Food type: Chlorella sp. suspension
- Amount: 0.25 ml (on weekends 0.50 mL), corresponding to 0.2 mg C/Daphnia/day
- Frequency: Twice daily (on weekends single dose)

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

161 - 179 mg CaCO3/liter

Test temperature:

19 - 21°C

pH:

7.6 - 8.4

Dissolved oxygen:

7.9 to 9.9 mg/L

Nominal and measured concentrations:

1.0 mg a.i./L

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass covered with a Perspex plate
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 60 ml (6 x Ø 3.5 cm)
- Renewal rate of test solution (frequency/flow rate): Three times a week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 20
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS: M7 (as in OECD 211)

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16h daily
- Light intensity: 512-630 lux at the start and 525-406 lux at the end of the exposure

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Parental daphnids
Condition: Every workday and upon renewal on non-workdays, 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 and upon renewal on non-workdays.
Body length: At the end of the test.
Offspring:
Appearance first brood: When observed.
Newborn daphnids: Every workday and upon renewal on non-workdays, the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.

TEST CONCENTRATIONS
A preliminary test was performed prior to the reproduction test. WAF’s prepared at a loading rate of 1.0, 10 and 100 mg a.i./L were tested. A control group was also included. Each concentration 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 including feeding. Test solutions were renewed on days 1, 3 and 6 during the test. Samples for possible analysis were taken at days 0 (fresh), 1 (fresh and old), 3 (fresh and old) and 6 (old). Samples were taken from solutions containing the daphnia and food (algae), but also from an extra vessel incubated under the same conditions, but without daphnia and algae. No significant mortality (>10%) was observed in the control and test concentrations. Two parental daphnids exposed to the WAF prepared at a loading rate of 10 mg a.i./L died during the ten-day test period. The presence of eggs in the brood pouch was recorded for the first time on day 5 for the control and all treated solutions. The first brood was observed on day 8. Therefore, no effect on time of egg development was observed in all treated solutions, when compared to the control. Based on the results of this test it was decided to perform the final test as a limit with the WAF prepared at a loading rate of 1.0 mg/L. This loading rate was chosen as it was observed that the test item was not completely dissolved at the end of the preparation procedure in this treatment, and thus the maximum solubility was reached. The loading rate of 1.0 mg/L is considered to be the saturation concentration.

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Details on results:

Mortality of parental Daphnids:
No parental daphnids died during the test period in the control.
Two parental daphnids died in the WAF prepared at a loading rate of 1.0 mg a.i./L
Hence, no significant mortality of the parental was observed in neither the control nor the treatment group, thus the NOEL for survival was 1.0 mg a.i./L.

Time to First Reproduction:
In the controls, the presence of eggs in the brood pouch was recorded for the first time on day 5 and the first brood appeared on day 8. The first recordings of eggs in the brood pouch and the first brood were exactly the same in the test item test group. Hence, no delay in appearance of the first brood was observed.

Reproduction:
On average, 167 offspring were produced per surviving daphnid in the control treatment whereas 172 offspring were counted on average in the limit group. Statistical analysis showed that the reproduction of the daphnids was not significantly different between the two test groups. Hence, the NOEL for reproduction was 1.0 mg a.i./L. There were a few recordings unhatched (aborted) eggs in both groups during the 21-day test period. These were considered to be insignificant and not test item related

Body Length:
There was no statistically significant difference between the mean body length of the parents exposed to the control and the limit concentrations. Hence, the NOEL for growth was 1.0 mg a.i./L.

Reported statistics and error estimates:

The following statistical procedures were used to determine the NOEC for reproduction and growth:
•Data distribution: Shapiro-Wilk´s Test
•Homogeneity of variance: Levene´s Test (with Residuals)
•Differences between treatment and the control: Student t-test for Homogeneous Variances (alfa=0.05, one-sided smaller).
Parental survival was analysed with Fisher`s Exact Binomial Test (alfa=0.05, one-sided greater).
No EC10 and EC50 values could be calculated as the observed effects were lower than 10%.
All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Mortality (immobility) of parental Daphnids:

Dose (mg a.i./L)	Introduced	Mobile	Immobile	% Immobility
Control	20	20	0	0
1.0	20	18	2	10

Group mean cumulative number of juveniles per surviving parent and reduction of reproduction:

Dose (mg a.i./L)	Mean	Std. Dev.	n	%Change
Control	167	18	20
1.0	172	11	18	-3.2

negative numbers indicate stimulation

Incidence of aborted eggs:

Dose (mg a.i./L)	Total offspring	Aborted eggs	%Aborted eggs
Control	3332	5	0.1
1.0	3324	1	0.0

Mean body lengths (mm) and reduction of growth of parental Daphnids

Dose (mg a.i./L)	Mean (mm)	Std. Dev.	n	% Reduction
Control	4.57	0.160	20
1.0	4.57	0.136	18	0.1

Validity criteria fulfilled:

yes

Remarks:

Mortality of parental in the control < 20% (0%); The average cumulative number of young/female in the control after 21 days was >60 (167 ± 11).

Conclusions:

Test item did not affect the reproduction of Daphnia magna at a loading rate of 1.0 mg a.i./L after 21 days of exposure, being the NOAEL equal to 1.0 mg a.i./L and EL50 greater than 1.0 mg a.i./L.

Executive summary:

A Daphnia magna reproduction test was performed according to the OECD Guideline 211 (GLP study). Based on preliminary test (10 -day range finding), a Water Accommodated Fraction (WAF) was prepared at 1.0 mg a.i./L and used as the limit concentration. The reproduction test was performed in a semi-static system, included 20 vessels for an untreated control group and 20 vessels for a WAF prepared at 1.0 mg a.i./L. 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 suspension. Every workday the condition of the parental daphnids was recorded, during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test the lengths of the surviving parental daphnids were measured. No mortality of the parental daphnids was observed at the untreated control while two parental daphnids died in the Water Accommodated Faction prepared at 1.0 mg a.i./L. On average, 167 offspring were produced per surviving daphnid in the control treatment whereas 172 offspring were counted on average in the limit concentration. There was no delay in first reproduction in the test group compared to the control. On day 8 an insignificant number of unhatched (aborted) eggs was observed in both test groups. The mean body length of the parents exposed to the control and the limit concentration were 4.6 mm, for both groups. The study met the acceptability criteria prescribed by the study plan and was considered valid. Based on these results, it can be concluded that test item did not affect the reproduction of Daphnia magna at a loading rate of 1.0 mg a.i./L after 21 days of exposure, being the NOAEL equal to 1.0 mg a.i./L and EL50 greater than 1.0 mg a.i./L.

Description of key information

Key study: OECD Guideline 211. GLP study. Test item did not affect the reproduction of Daphnia magna at a loading rate of 1.0 mg a.i./L after 21 days of exposure, being the NOAEL equal to 1.0 mg a.i./L and EL50 greater than 1.0 mg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 mg/L