Isobutyl 2-naphthyl 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:

22 July 2016 - 09 September 2016

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:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

2.0 mL samples from all test concentrations and the control (taken from approximate centre of the test vessels) at the start and the end of both 24-hour renewal periods. At the end of each renewal period, samples were taken from one replicate of each test concentration and the control (replicates were not pooled).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
The test item was a white solid and was heated to approximately 60°C during a period of about one hour prior to weighing. During this time, the test item turned into a clear liquid. No correction was made for the composition of the test item. Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring with minimum headspace followed by a settlement period of maximal 3.5 hours. Afterwards, the Saturated Solution (SS) was siphoned off and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless. The controls contained test medium without test item or other additives.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magnia (Straus, 1820)
- Source: In-house laboratory culture with a known history, 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.
- Age at study initiation: young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- 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 medium (M7) in an all-glass culture vessel. Cultures were renewed after 7 days of cultivation, half of the medium twice a week.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

180 mg/L (as CaCO3)

Test temperature:

20 - 21 °C

pH:

8.0-8.1 (in fresh and old solutions)

Dissolved oxygen:

8.6 - 9.2 mg/L (fresh solutions)
8.5 - 9.0 mg/L (old solutions)

Nominal and measured concentrations:

- Nominal concentrations: 0.46, 1.0, 2.2, 4.6 and 10 % of a saturated solution (SS) prepared at a loading rate of 100 mg/L (based on the results of the combined limit/range test).
- Average measured exposure concentrations: 0.10, 0.18, 0.39, 0.72 and 1.3 mg/L

Effect parameters were determined using geometric mean measured concentrations (see 'Any other information on materials and methods incl. tables').

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, minimum headspace, closed airtight, fil volume: 60 mL
- Aeration: no
- Renewal rate of test solution: after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: adjusted ISO medium
- Culture medium different from test medium: yes, M7

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h daily

EFFECT PARAMETERS MEASURED:
Mobility at 24 and 48 hours

PHYSICO-CHEMICAL APAREMETERS MEASURED
pH and dissolved oxygen were measured at the start and the end of both renewal periods, for all concentrations and the control; temperature of medium was measured continuously in a temperature control vessel, beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (July 2016)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 0.27 - 0.41 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.89 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 0.74 - 1.0 mg/L

Details on results:

- Behavioural abnormalities: trapping at surface of test solutions was observed in all test levels (but not all replicates of 0.10, 0.39, 0.72 and 1.3 mg/L, all replicates of 0.18 mg/L) after 24 hours, and in one replicate of 0.10 mg/L and all replicates of 0.18 and 0.39 mg/L test solutions after 48 hours (see table 3 below)
- Other biological observations: none reported
- Immobility of control: yes, 10% immobilized at 24 and 48 hours
- Other adverse effects control: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

48-EC50: 0.39 mg/L (95%-CI: 0.33 - 0.44 mg/L)

Reported statistics and error estimates:

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

For details on measured concentrations per time period, see table 1 below. All concentrations decreased slightly to 58-95% of initial at the end of the first 24-hour renewal period. Measured concentrations at the start of the second renewal period were 0.071, 0.38, 0.65, 1.4 and 2.7 mg/L in 0.46, 1.0, 2.2, 4.6 and 10% of a SS prepared at 100 mg/L, respectively. The four highest test concentrations decreased to 50-79% of initial at the end of the test, while in the lowest test concentration an unexpected high 0.37 mg/L was measured (522% relative to the analysed value in the freshly prepared solution). These results indicated that the preparation of the SS was not repeatable and that the decrease of concentrations during the 24-hour renewal periods was not consistent over the tested concentration range. Given this result, the EC50 values were expressed in terms of the average measured exposure concentrations (see Table 2). The range tested based on the average measured concentrations was 0.10, 0.18, 0.39, 0.72 and 1.3 mg/L.

Because of the unexpected high measured concentration of 0.37 mg/L at t=48, it was decided to analyse the reserve samples. Analysis of the reserve samples taken at the end of the second renewal period was consistent with the measured concentrations in the main set of samples, except for the lowest concentration where now a lower concentration of 0.13 mg/L was measured. The mean of the two measurements was used at t=48 hours.

Table 1          Concentrations of the test item in test medium - final test

Time of sampling ^1 [hours]	Percentage of SS^2 [%]	Analysed concentration [mg/L]	Relative to initial [%]
0 (fresh)	0	n.d.
	0.46	0.0860
	1.0	0.105
	2.2	0.182
	4.6	0.402
	10	0.823
24 (old)	0	n.d.	n.a.
	0.46	0.0533	62
	1.0	0.0607	58
	2.2	0.169	93
	4.6	0.382	95
	10	0.686	83
24 (fresh)	0	n.d.
	0.46	0.0709
	1.0	0.375
	2.2	0.653
	4.6	1.38
	10	2.68
48 (old)	0	n.d.	n.a.
	0.46	0.370	522*
	1.0	0.217	58
	2.2	0.516	79
	4.6	0.815	59
	10	1.33	50
48 (old)^3	0	n.d.	n.a.
	0.46	0.133	187
	1.0	0.216	58
	2.2	0.627	96
	4.6	0.798	58
	10	1.34	50

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

²      Percentage of a saturated solution prepared at a loading rate of 100 mg/L.

³      Reserve samples.

n.d. Not detected. The limit of detection of the method was determined to be 0.0021 mg/L taking a dilution factor of two into account.

n.a. Not applicable.

* After a second sample the measuered 522% showed to be 187% and more in line with the other measured concentrations.

  Table 2        Average exposure concentration versus nominal concentration

Naph Iso Butyl Ether Beta % SS prep. at 100 mg/L	Measured concentration (mg/L)						Average exposure concentration (mg/L)
	t=0h (fresh)	t=24h (old)	t=24h (fresh)	t=48h (old)	t=48h (old - reserve)	t=48h (old - average)
0.46	0.0860	0.0533	0.0709	0.370	0.133	0.25	0.10
1.0	0.105	0.0607	0.375	0.217	0.216	0.22	0.18
2.2	0.182	0.169	0.653	0.516	0.627	0.57	0.39
4.6	0.402	0.382	1.38	0.815	0.798	0.81	0.72
10	0.823	0.686	2.68	1.33	1.34	1.3	1.3

Table 3       Number of introduced daphnids and incidence of immobility in the final test

Time (h)	Replicate	Average concentration Naph Iso Butyl Ether Beta (mg/L)
		Control	0.10	0.18	0.39	0.72	1.3
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 (1)	0 (2)	0	3 (4)	5 (1)
	B	0	0	0 (1)	0	1 (2)	5 (1)
	C	1	0 (1)	0 (2)	0 (2)	0	4
	D	1	0 (1)	0 (1)	1 (1)	2 (1)	4
	Total immobilised	2	0	0	1	6	18
	Effect %	10	0	0	5	30	90
48	A	0	1	0 (2)	2 (2)	5	5
	B	0	0	0 (3)	2 (2)	5	5
	C	1	0	2 (1)	3 (3)	5	5
	D	1	1	1 (2)	3 (1)	5	5
	Total immobilised	2	2	3	10	20	20
	Effect %	10	10	15	50	100	100

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

Validity criteria fulfilled:

yes

Remarks:

The criteria as presented in See 'Any other information on materials and methods incl. tables' are fulfilled.

Conclusions:

The 48h-EC50 value to Daphnia magna was 0.34 mg/L based on geometric mean concentrations.

Executive summary:

Based on the results of a combined limit/range-finding test, the following nominal concentrations were used in the definitive test: 0.46, 1.0, 2.2, 4.6 and 10% of a saturated solution prepared at a loading rate of 100 mg/L. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24-hour renewal periods were analysed with a validated UPLC method. The maximum water solubility was 0.8 - 2.7 mg/l and the (dilutions of the) concentrations in the saturated solutions were not fully repeatable. At the end of the 24 hours renewal period the measured concentration were ca 50-95% of the initial measured ones. Therefore the final concentrations tested were based on geometric mean measured concentrations: 0.10, 0.18, 0.39, 0.72 and 1.3 mg/L. The 48h-EC50 value to Daphnia magna was 0.34 mg/L based on geometric mean measured concentrations.

Description of key information

Based on the results of a combined limit/range-finding test, the following nominal concentrations were used in the definitive test: 0.46, 1.0, 2.2, 4.6 and 10% of a saturated solution prepared at a loading rate of 100 mg/L. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24-hour renewal periods were analysed with a validated UPLC method. The maximum water solubility was 0.8 - 2.7 mg/l and the (dilutions of the) concentrations in the saturated solutions were not fully repeatable. At the end of the 24 hours renewal period the measured concentration were ca 50-95% of the initial measured ones. Therefore the final concentrations tested were based on geometric mean measured concentrations: 0.10, 0.18, 0.39, 0.72 and 1.3 mg/L. The 48h-EC50 value to Daphnia magna was 0.34 mg/L based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.34 mg/L