Reaction mass of octan-2-ylbenzene, octan-3-ylbenzene and octan-4-ylbenzene

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:

05 july - 20 Aug 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. Moreover, in the definitive test, samples of all stock solutions were also taken.
Frequency:
at t=0 and 24 h from freshly prepared test solutions
At 24 and t=48 h from spent test solutions
At t=-2 and -1 day, from stock solutions

Volume:
10 mL from the approximate centre of the test vessels
3.0 mL from each stock solution

Storage:
Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

Remarks:

Hexane was evapurated before adding the test medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Combined limit/range-finding test and two full tests:
Preparation of test solutions started with individually prepared loading rates ranging from 0.46 to 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of two hours. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by means of siphoning.

Definitive test:
A stock solution of 6 g/L was prepared in hexane (Merck, Darmstadt, Germany). Subsequently, stock solutions of 0.57, 1.0, 1.9, 3.3 and 6.0 mg/L were prepared by a series of dilutions in hexane. Fresh stock solutions were prepared for each renewal of test solutions. Test solutions used for the exposure were prepared as follows:
0.5 mL of a respective stock solution was added to a 100 mL Erlenmeyer flask. Hexane was allowed to evaporate for a period of 2 hours after which 50 mL of test medium was added to each vessel. A two-day period of shaking was applied to ensure maximum dissolution of the test item in test medium. The obtained Water Accommodated Fractions (WAFs) were clear and colorless.

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
- Method of breeding: Start of each batch with approximately 250 newborn daphnids, i.e. less than 3 days old, by placing them into 5 litres of M7 medium in an all-glass culture vessel.
Maximum age of the culture:4 weeks
Renewal of the culture: After 7 days of cultivation, half of the medium twice a week.
Feeding: Daily, a suspension of fresh water algae
- Validity of the 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.
- Age at Study Initiation: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.
- Feeding during test : No feeding

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:

between 19 and 21°C

pH:

7.9 - 8.3

Dissolved oxygen:

8.9 - 9.8 mg/L

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Definitive test:
Nominal concentrations:WAFs prepared at loading rates of 5.7, 10, 19, 33 and 60 µg/L.

Measured concentrations: Samples taken from the stock prepared at 6.0 mg/L, blank hexane, the highest test concentration and the control were analysed.
The concentrations measured in the test solutions were below the level of quantification of the analytical method, i.e. below 0.01 µg/L.
No test item was observed in hexane used for preparation of stock solution and preparation of the control solution. Concentrations measured in the stock solution of 6.0 mg/L were 6.7 and 4.7 mg/L (112 and 78% of nominal) upon the first and the second renewal, respectively. Based on these results, it was assumed that test concentrations were prepared correctly. However, exact exposure concentrations could not be provided due to the difficult nature of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass.
- Volume of solution: 50 mL
- Aeration: No aeration of the test solutions was applied.
- 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: 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
- Culture medium different from test medium: yes, for culturing M7 medium is used.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A daily photoperiod of 16 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 24h-EC50, 48h-EC50

RANGE-FINDING STUDY
- Test concentrations: WSFs prepared at loading rates of 1.0, 10, and 100 mg/L.

FIRST FULL TEST
- Test concentrations: WSF's prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L

SECOND FULL TEST
- Testconcentrations: WSF's prepared at loading rates of: 0.46, 1.0, 2.2, 10, 22 and 46 mg/L
- Measured concentrations:
Start of the test: 0.065, 0.078, 0.082, 0.082, 0.11. 0.11 and 0.14 mg/L
End of the test: 0.0051, 0.0054, 0.0047, 0.0076, 0.0069, 0.0042 and 0.0019 mg/L
Average expose conc.: 0.018, 0.020, 0.020, 0.025, 0.028, 0.021 and 0.016 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7,)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 60 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: the 48h-EC50 for Daphnia magna exposed to SHR 1396 exceeded a WAF loading rate of 60 µg/L, i.e. was >40 µg/L, and thus exceeded the water solubility limit of the test item.

Details on results:

Second Full Test:

- No immobility was observed in the control throughout the exposure period. Similar to the first full test, no immobility was observed at 24 hours of exposure at any of the concentrations
tested. At the end of the test, immobilisation was ≥ 50% in all groups correlating to the appliedloading rates.
- Measured concentrations: Initial: 0.065, 0.078, 0.082, 0.082, 0.11, 0.11 and 0.14 mg/L. Eend of the test: 0.0051, 0.0 54, 0.0047. 0.0076, 0.0069, 0.0042, 0.0019 mg/L. Average sxposure: 0.018, 0.020, 0.020, 0.025, 0.028, 0.021, 0.016 mg/L.
- The 24h-EC50 was > 140 μg/L and the 48h-EC50 was 65 μg/L based on intial measured concentrations. It should be noted that the water solubility of the test item was determined to be 0.033 mg/L (Test Facility Study No. 20197453), hence the calculated EC50 exceeded the water solubility of the test item.
- The OECD 23 guideline recommends using the average exposure concentrations for the calculation of effect parameters in case the measured concentrations decrease by more than
20% during the exposure. However, in this study no relation was observed between the average exposure concentrations and the immobilisation rate.

The exposure concentrations in the combined limit/range-finding and first two full tests substantially exceeded the solubility limit of test item in water and most probably also in test medium, although the latter is unknown. This is substantiated by the fact that the measured concentrations showed a rapid decline during the test duration. For this reason, effect parameters obtained in these tests are considered to not be representative.

Definitieve Test:

In the definitive test, care was taken not to produce oversaturated solutions (by spiking at and below water solubility) and to keep the concentrations as stable as possible by renewing the test solutions after 24 hours of exposure. The effect parameters obtained in this test are considered reliable and representative.

- Immobility of control: No immobility was observed in the control throughout the exposure period
- Other adverse effect control:no
- Effect concentrations exceeding solubility of substance in test medium:yes

Results with reference substance (positive control):

- Results with reference substance valid: The study generally met the acceptability criteria prescribed by the study plan and was considered valid
- Dose-response test: concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L.
- ECx:
The 24h-EC50 was 0.52 mg/L with a 95% confidence interval between 0.45 and 0.58 mg/L.
The 48h-EC50 was 0.33 mg/L with a 95% confidence interval between 0.27 and 0.38 mg/L

SECOND FULL TEST

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

 

Time (h)	Replicate	SHR 1396; WSF prepared at a loading rate (mg\L)
		Control	0.46	1.0	2.2	4.6	10	22	46
0	A	5	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20	20
24	A	0	0	0	0	0	0	0	0
	B	0	0	0	0	0	0	0	0
	C	0	0	0	0	0	0	0	0
	D	0	0	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0	0	0
48	A	0	2	2	4	3	4	5	4
	B	0	1	5	4	3	3	5	5 (1)
	C	0	3	2	4	3	4	3	5
	D	0	4^#	2	4	3	3	5	5
	Total immobilised	0	10	11	16	12	14	18	19
	Effect %	0	50	55	80	60	70	90	95

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. This organism was reimmersed into the respective solutions before recording of mobility.^#Microscopic observation revealed no test item attached to the daphnids.

Table 2:

Average Exposure Concentration Versus Nominal Loading Rates in theSecond Full Test

 

SHR 1396 WSF loading rate (mg/L)	Measured concentrations (mg/L)		Average exposure conc. (mg/L)
	t=0h	t=48h
0.46	0.065	0.0051	0.018
1.0	0.078	0.0054	0.020
2.2	0.082	0.0047	0.020
4.6	0.082	0.0076	0.025
10	0.11	0.0069	0.028
22	0.11	0.0042	0.021
46	0.14	0.0019	0.016

DEFINITIVE TEST:

Table 3:

Number of Introduced Daphnids and Incidence of Immobility in the Definitive Test

 

Time (h)	Replicate	SHR 1396; Nominal concentration(µg/L)
		Control	5.7	10	19	33	60
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	6 1	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	21	20	20	20	20	20
24	A	0	0	0	0	0	0
	B	0	0	0	0	0	0
	C	0	0	0	0	0	0
	D	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0
48	A	0	0	0	1	0	0
	B	0	0	0	0	0	0
	C	0	0	0	0	0	0
	D	0	0	0	0	0	0
	Total immobilised	0	0	0	1	0	0
	Effect %	0	0	0	5	0	0

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to SHR 1396 exceeded a WAF loading rate of 60 µg/L, i.e. was >40 µg/L, and thus exceeded the water solubility limit of the test item.

Executive summary:

The short-term toxicity of the test item to aquatic invertebrates was studies in a daphnia acute study according to OECD guideline No. 202. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23.

A definitive test was performed based on the results of a combined limit/range-finding test and two full tests. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAF's prepared at loading rates of 5.7, 10, 19, 33 and 60 µg/L. Samples of the freshly prepared test solutions were taken at the start of the test and at the 24 hours renewal. Samples of the spent test solutions were taken at 24h and at the end of the test period. Samples of the stock solutions in hexane were taken from the freshly prepared stock solutions.

Samples taken from the stock prepared at 6.0 mg/L, blank hexane, the highest test concentration and the control were analysed.

The concentrations measured in the test solutions were below the level of quantification of the analytical method, i.e. below 0.01 µg/L.

No test item was observed in hexane used for preparation of stock solution and preparation of the control solution. Concentrations measured in the stock solution of 6.0 mg/L were 6.7 and 4.7 mg/L (112 and 78% of nominal) upon the first and the second renewal, respectively. Based on these results, it was assumed that test concentrations were prepared correctly. However, exact exposure concentrations could not be provided due to the difficult nature of the test item.

No significant immobilisation (i.e. >10%) was observed at any concentration tested and in the control during the exposure period.

In conclusion, the 48h-EC₅₀ for Daphnia magna exposed to SHR 1396 exceeded a WAF loading rate of 60 µg/L, i.e. was >40 µg/L, and thus exceeded the water solubility limit of the test item.

Description of key information

In conclusion, the 48h-EC₅₀ for Daphnia magna exposed to SHR 1396 exceeded a WAF loading rate of 60 µg/L, i.e. was >40 µg/L, and thus exceeded the water solubility limit of the test item.

Additional information

The short-term toxicity of the test item to aquatic invertebrates was studied in a daphnia acute study according to OECD guideline No. 202. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23.

A definitive test was performed based on the results of a combined limit/range-finding test and two full tests. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAF's prepared at loading rates of 5.7, 10, 19, 33 and 60 µg/L. Samples of the freshly prepared test solutions were taken at the start of the test and at the 24 hours renewal. Samples of the spent test solutions were taken at 24h and at the end of the test period. Samples of the stock solutions in hexane were taken from the freshly prepared stock solutions.

Samples taken from the stock prepared at 6.0 mg/L, blank hexane, the highest test concentration and the control were analysed.

The concentrations measured in the test solutions were below the level of quantification of the analytical method, i.e. below 0.01 µg/L.

No test item was observed in hexane used for preparation of stock solution and preparation of the control solution. Concentrations measured in the stock solution of 6.0 mg/L were 6.7 and 4.7 mg/L (112 and 78% of nominal) upon the first and the second renewal, respectively. Based on these results, it was assumed that test concentrations were prepared correctly. However, exact exposure concentrations could not be provided due to the difficult nature of the test item.

No significant immobilisation (i.e. >10%) was observed at any concentration tested and in the control during the exposure period.

In conclusion, the 48h-EC₅₀for Daphnia magna exposed to SHR 1396 exceeded a WAF loading rate of 60 µg/L, i.e. was >40 µg/L, and thus exceeded the water solubility limit of the test item.