C12-16 alkyl, glycerol ether

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct 2018 - 28 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

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:

2018

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: C12 – 16 alkyletherdiol
Appearance: Opaque white to light yellow liquid (determined by Charles River Den Bosch)
CAS: Not available
Lot/Batch: D7801-171116001
Date of Production: 17.11.2017
Best before Date: Within 2 years
UVCB 100%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method: 2.0 mL from the approximate centre of the test vessels; at the start of the test and after 24 h from the freshly prepared solutions, at the renewal (t=24h) and the end of the test from the 24-h old solutions.
- Sample storage conditions before analysis: in a freezer (≤-15°C) at the analytical laboratory of the Test Facility
- At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with loading rates individually ranging between 0.46 and 100 mg/L. A 2-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. 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, with the exception of the WAF prepared at a loading rate of 100 mg/L in the combined limit/range-finding test which was slightly hazy and colorless. All test solutions were checked for the Tyndall effect using a laser pen. No light scattering was observed.
- Controls: Test medium without test item or other additives.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Source: In-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: > two weeks old
- Age of test organisms at test start: < 24 hours
- Feeding during test: no
- 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.
- Daphnids were introduced to the test solutions within 18 minutes after preparation of the test solutions.

BREEDING
- Method of breeding: start of each batch with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 medium in an all-glass culture vessel.
- Feeding: Daily, a suspension of fresh water algae
- Temperature of medium: 18-22°C
- Renewal of the cultures: after 7 days of cultivation half of the medium twice a week.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L (expressed as CaCO3)

Test temperature:

18 - 20°C

pH:

t=0 h: 7.9 - 8.0
t=24 h, old: 7.8
t=24 h, fresh: 7.7 - 7.8
t=48 h: 7.9

Dissolved oxygen:

t=0 h: 9.1 - 9.4 mg/L
t=24 h, old: 9.9 - 10 mg/L
t=24 h, fresh: 9.9 mg/L
t=48 h: 9.0 - 9.2 mg/L

Nominal and measured concentrations:

Nominal: WAFs individually prepared at loading rates of 0.47, 1.0, 2.2, 4.6 and 10 mg/L. The lowest loading was targeted at 0.46 mg/L, but was slightly higher (0.49 mg/L) at the start of the test. At the renewal, nominal loading rate was 0.46 mg/L. Average loading rate was therefore 0.47 mg/L. This was considered acceptable since the effect parameters were based on the actual measured concentrations.
Measured concentrations: Concentrations measured in the freshly prepared solutions were below the LOD in the two lowest groups, while a dose related increase was observed at higher test concentrations. Measured concentrations decreased by more than 27% during both refreshments. Averaged geometric mean measured exposure concentrations were calculated to be 0.00039, 0.00039, 0.55, 2.0 and 4.2 mg/L. See 'Any other information on materials and methods' for calculation of average exposure concentrations and 'Any other information on results' for details on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, fill volume: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Medium: Adjusted ISO medium.
- Culture medium different from test medium: yes, M7 medium
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning and at the end of the test, for all concenctrations and the control; Temperature of the medium: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours per day

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Immobility (including mortality): at 24 and 48 hours.

TEST CONCENTRATIONS
- Combined limit/range-finding test: yes, with WAFs individually prepared at loeading rates of 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: yes, EC50 was expected to be between concentrations reached in WAFs prepared at loading rates of 1.0 and 10 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Nov 2018)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.66 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 0.44 - 0.99 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit: 0.93 - 1.6 mg/L

Details on results:

- Behavioural abnormalities: none observed in control
- No immobility was observed in the control and at the two lowest concentrations tested throughout the exposure period. At the end of the test, 30% immobility was observed at an average exposure concentration of 0.55 mg, while total immobility was observed at the two highest test concentrations.
- The measured concentrations at the start of the test were below the limit of detection in the two lowest WAFs tested, while they were 0.64, 2.6 and 4.8 mg/L in WAFs prepared at loading rates of 2.2, 4.6 and 10 mg/L. The measured concentrations decreased below the limit of detection in the WAF prepared at loading rate of 2.2 mg/L, and to 0.63 and 2.3 mg/L at the two highest WAFs tested, respectively, at the end of the first renewal. At the start of the second renewal period, the measured concentrations were below the limit of detection in the two lowest WAFs tested, while they were 1.3, 3.4 and 6.0 mg/L in WAFs prepared at loading rates of 2.2, 4.6 and 10 mg/L, respectively. At the end of the test, the measured concentrations decreased to 0.86, 2.3 and 4.4 mg/L, respectively.
A response below the limit of detection was observed in all controls throughout the test, which was at the carry-over level of the analytical system. Where concentrations were below the level of detection (i.e., at the level of the carry-over), it could not be ascertained whether this concentration originated from the test sample or the analytical system. However, since these concentrations were estimated as half of the limit of detection, the worst-case assumption has been followed.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 48-h EC50: 0.40 mg/L, 95% confidence interval 0.33 - 0.48 mg/L
- The actual responses in this reference test with K2Cr2O7 are generally within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.28 and 0.90 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility

Reported statistics and error estimates:

The 24 and 48h-EC50-values were calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test item average exposure concentrations using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Final Test: Test Samples Analysis Results

Time of sampling [hours]	Loading rate (a) [mg/L]	Concentration analyzed [mg/L]	Relative to initial [%]
0	0	< LOD (b)
(fresh)	0.47	< LOD (b)
	1.0	< LOD (b)
	2.2	0.635
	4.6	2.55
	10	4.75
24	0	< LOD (b)	n.a.
(old)	0.47	< LOD (b)	n.a.
	1.0	< LOD (b)	n.a.
	2.2	< LOD (b)	n.a.
	4.6	0.629	25
	10	2.25	47
24	0	< LOD (b)
(fresh)	0.47	< LOD (b)
	1.0	< LOD (b)
	2.2	1.34 (c)
	4.6	3.35
	10	6.04
48	0	< LOD (b)	n.a.
(old)	0.47	< LOD (b)	n.a.
	1.0	< LOD (b)	n.a.
	2.2	0.863	64
	4.6	2.32 (c)	69
	10	4.42 (c)	73

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

(b) The limit of detection (LOD) of the analytical method was determined to be 0.00078 mg/L taking a dilution factor of two into account.

(c) Concentrations were calculated by extrapolation of the calibration curve. Hence these samples were re-diluted and analyzed the next day.

n.a.  Not applicable.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the Final Test 

Time (h)	Replicate	Average exposure conc. (mg/L)
		Control	0.00039	0.00039	0.55	2.0	4.2
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	5^#	5
	B	0	0	0	0	4	5
	C	0	0	0	0	5	5
	D	0	0	0	0	4	5
	Total immobilised	0	0	0	0	18	20
	Effect %	0	0	0	0	90	100
48	A	0	0	0	1	5	5
	B	0	0	0	2	5	5
	C	0	0	0	2	5	5
	D	0	0	0	1	5	5
	Total immobilised	0	0	0	6	20	20
	Effect %	0	0	0	30	100	100

^#Microscopic observation revealed no test item attached to the daphnids.

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 48h-EC50 of the substance to Dapnhia magna was determined to be 0.66 mg/L, based on average exposure concentrations (95% confidence interval 0.44 - 0.99 mg/L).

Executive summary:

In a semi-static 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to Water Accommodated Fractions (WAFs) of the test substance individually prepared at loading rates of 0.47, 1.0, 2.2, 4.6 an 10 mg/L and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). Concentrations measured in the freshly prepared solutions were below LOD in the two lowest treatments, while a dose related increase was observed at the higher test concentrations. Measured concentrations decreased by more than 27% during both refreshments. Therefore, averaged geometric mean measured exposure concentrations were used to determine the effect parameters and were calculated to be 0.00039, 0.00039, 0.55, 2.0 and 4.2 mg/L.

No immobility was observed in the control and the two lowest test concentrations throughout the exposure period. At the end of the test, 30% immobility was observed at 0.55 mg/L, while total immobility was observed at the two highest test concentrations.

The 48h-EC50 for immobility was 0.66 mg/L based on average exposure concentrations (95% confidence interval between 0.44 and 0.99 mg/L).

The study met all validity criteria and is considered to be reliable without restrictions.

Description of key information

The 48h-EC50 of the substance to Dapnhia magna was determined to be 0.66 mg/L, based on average exposure concentrations (95% confidence interval 0.44 - 0.99 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.66 mg/L