Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with Phytosterol, Isooctadecan-1-ol, Hexadecan-1-ol, Octadecan-1-ol and Docosan-1-ol

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 - 24 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23

Version / remarks:

2000

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with Phytosterol, Isooctadecan-1-ol, Hexadecan-1-ol, Octadecan-1-ol and Docosan-1-ol

EC Number:

817-761-7

Cas Number:

765293-81-0

Molecular formula:

Not applicable

IUPAC Name:

Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with Phytosterol, Isooctadecan-1-ol, Hexadecan-1-ol, Octadecan-1-ol and Docosan-1-ol

Test material form:

other: waxy paste

Details on test material:

Appearance: White to pale yellow waxy paste
Test item storage: At room temperature
Stable under storage conditions until: 30 May 2019 (retest date)

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of Test Solutions
The batch of Plandool-H tested was a white to pale yellow waxy paste UVCB and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A two-day period of magnetic stirring at 45°C was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight (approximately a period of 22 hours). Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
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 or discoloured animals and there was no delay in the production of the first brood.
Characteristics: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Breeding
Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Culture 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).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands): CaCl2.2H2O: 211.5 mg/L; MgSO4.7H2O: 88.8 mg/L; NaHCO3: 46.7 mg/L; KCl: 4.2 mg/L.
The hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

19 - 20°C

pH:

7.9 - 8.2

Dissolved oxygen:

7.3 - 8.9 mg/L

Details on test conditions:

Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass
Test medium: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands): CaCl2.2H2O: 211.5 mg/L; MgSO4.7H2O: 88.8 mg/L; NaHCO3: 46.7 mg/L; KCl: 4.2 mg/L.
Number of daphnids: Control and highest concentration: 20 per test group; Intermediate concentrations: 10 per concentration.
Loading: 5 per vessel containing 50 mL of test solution.
Light: A daily photoperiod of 16 hours.
Feeding: No feeding
Aeration: No aeration of the test solutions was applied.
Introduction of daphnids: 17 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (tested periodically)

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: the effect based on loading rate

Details on results:

No biologically relevant immobility was observed in the control and at any of the test concentrations throughout the exposure period.
In conclusion, the 48h-EL50 for Daphnia magna exposed to Plandool-H exceeded the maximum solubility of the test item in test medium, i.e. exceeded a loading rate of 100 mg/L.

Results with reference substance (positive control):

The 24h-EC50 in this reference test with K2Cr2O7 was within the expected range of 0.6 and 2.1 mg/L as specified in ISO International Standard 6341, October 2012. The 48h-EC50 was within the expected range of 0.28 and 0.75 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
The 24h-EC50 was 0.84 mg/L with a 95% confidence interval between 0.73 and 0.97 mg/L.
The 48h-EC50 was 0.47 mg/L with a 95% confidence interval between 0.41 and 0.55 mg/L
In conclusion, the sensitivity of this batch of Daphnia magna was in agreement with ISO International Standard 6341, October 2012 and the historical data collected at Charles River Den Bosch.
Period of reference test: 21 - 23 Jan 2019

Any other information on results incl. tables

ANALYSIS

No EL50-values could be calculated because the observed effects were below 50% (EL50 > maximum soluble concentration tested)

Measured Concentrations

The results of analysis of the samples taken during the test are described in the following table:

Time of sampling [hours]	Date of sampling	Date of analysis^1	Loading rate^2[mg/L]	Concentration analyzed [mg/L]	Relative to initial [%]
0	17 Dec 2018	24 Dec 2018	0 100	^3 n.d.
48	19 Dec 2018	24 Dec 2018	0 100	n.d. n.d.	n.a. n.a.

¹Samples were stored in the freezer (≤-15°C) until the day of analysis.

²A water accommodated fraction (WAF) prepared at the loading rate.

³A small response was observed but the concentration was lower than the determined limit of detection of 0.078 mg/L taking a dilution factor of 2 into account. This was considered caused by carry over, as a similar response was observed in the preceding analytical blank.

n.d. Not detected. The limit of detection of the analytical method was determined to be 0.078 mg/L taking a dilution factor of two into account.

n.a. Not applicable.

Samples taken from the highest test concentration and the control were analysed. No test item was detected throughout the test at the highest concentration tested.

It should be noted that a very small response was measured in the control sample at the start of the test, which was caused by carry-over. Since not more than 10% of the daphnids showed signs of disease or stress (i.e. 10%) and this response was caused by carry-over, this small response has no effect on the study results or conclusions.

Based on these results and by decision of the sponsor, effect parameters were reported and expressed as loading rates.

Immobility

Table 1 shows the responses recorded during the test.

No biologically relevant immobility was observed in the control and at any of the test concentrations throughout the exposure period. It should be noted that ≤10% immobility or entrapment is allowed in the control, and therefore effects <10% were considered biologically not relevant.

The responses recorded in this test allowed for reliable determination of an EL₅₀.

Table 1 Number of Introduced Daphnids and Incidence of Immobility in the Test

Time (h)	Replicate	Control	Plandool-H Loading rate 1.0 mg/L	Plandool-H Loading rate 10 mg/L	Plandool-H Loading rate 100 mg/L
0	A	5	5	5	5
0	B	5	5	5	5
0	C	5			5
0	D	5			5
0	Total introduced	20	10	10	20
24	A	0	1	0	0
24	B	0	0	0	0
24	C	0			0
24	D	0 (2)			0
24	Total immobilized Effect %	0 0	1 5	0 0	0 0
48	A	0	0	0	0
48	B	0	0	0	0
48	C	0			0
48	D	0			0
48	Total immobilized Effect %	0 0	0 0	0 0	0 0

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

Experimental Conditions

The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 3. These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the test).

The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within ±1°C).

Table 3 pH and Oxygen Concentrations (mg/L) During the Test

Plandool-H Loading rate (mg/L)	Start (t=0 h) O2	Start (t=0 h) pH	End (t=48 h) O2	End (t=48 h) pH
Control	8.9	7.9	8.9	8.1
100	7.3	8.2	8.5	8.1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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