Heptanal, oligomeric reaction products with aniline

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:

29 October 2016 to 24 November 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

GLP compliance:

yes

Specific details on test material used for the study:

Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
pH (1% in water, indicative range): 8.0 – 7.8 (determined by Charles River Den Bosch)
Specific gravity/density: 0.9130

Analytical monitoring:

yes

Details on sampling:

Sampling for analysis of test concentrations
Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The glass wool was also retained for possible analyses.

Frequency: at t=0 h and t=48 h
Volume: 2.0 mL from the approximate centre of the test vessels
Storage : Not applicable, samples were analysed on the day of sampling.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Vehicle:

yes

Details on test solutions:

Preparation of test solutions
The batch of Hepteen Base® tested was a clear amber liquid with a purity of 99.7% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring, after which the resulting mixture was allowed to stabilize for approximately 2 hours. Subsequently, the Saturated Solution (SS) was siphoned off through glass wool 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.

Test organisms (species):

Daphnia magna

Details on test organisms:

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.

Reason for selection
This system has been selected as an internationally accepted invertebrate species.

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
For the test young daphnids were selected with an age of < 24 hours from parental daphnids older than two weeks.

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 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.

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).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3.

Test temperature:

18-22°C

pH:

pH: 6-9, not varying by more than 1.5 units

Dissolved oxygen:

≥3 mg/L

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Measured concentrations of 0.071, 0.14, 0.37, 0.67 and 1.4 mg/L in solutions containing 0.46, 1.0, 2.2, 4.6 and 10% of a saturated solution prepared at a loading rate of 100 mg/L

Details on test conditions:

Test procedure and conditions
Test duration: 48 hours
Test type: Static
Test vessels: 100 mL, all-glass
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 80 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 31 minutes after preparation of the test solutions.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%-confidence interval (mg/L) 0.18-0.19

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%-confidence interval (mg/L) 0.15-0.72

Details on results:

Combined limit/range-finding test
No biological significant number of daphnids became immobilize in the control and at the lowest test concentration throughout the test duration , while effects were complete in the two highest test concentrations already after 24 hours of exposure. The 48h-EC50 was expected to be between solutions containing 1.0% and 10% of a SS prepared at a loading rate of 100 mg/L.

Microscopic observations of daphnids exposed to solutions containing 1.0% and 10% of the SS prepared at loading rate of 100 mg/L at 24 hours of exposure revealed no test item attached to their bodies.

Samples taken from all concentrations were analysed. The actual concentrations were 0.034, 0.70 and 7.4 at the start of the test in 1.0, 10 and 100% of a SS prepared at a loading rate of 100 mg/L, respectively. Measured concentrations did not decrease by more than 20% of the initial concentrations throughout the test duration (86-99% of initially measured concentrations).

All test conditions were maintained within the limits prescribed by the study plan.

Final test-Measured concentrations
Analysis of the samples taken at the start (t=0) of the final test showed measured concentrations of 0.071, 0.14, 0.37, 0.67 and 1.4 mg/L in solutions containing 0.46, 1.0, 2.2, 4.6 and 10% of a SS prepared at a loading rate of 100 mg/L, respectively. During the exposure period the measured concentrations had decreased to 0.037, 0.081, 0.21, 0.39 and 0.90 mg/L, respectively.

Based on these results, the average exposure concentrations were calculated. The range tested based on average measured concentrations was 0.051, 0.11, 0.28, 0.51 and 1.1 mg/L.

Immobility
After 24 hours of exposure, no immobility was recorded in the control and at the two lowest test concentrations, while 50%, 90% and 95% daphnids were immobilized at average exposure concentrations of 0.28 mg/L, 0.51 mg/L and 1.1 mg/L, respectively. At the end of the exposure period, no immobility was recorded in the lowest test concentration, while 20% of daphnids were immobilized at an average exposure concentration of 0.11 mg/L and all daphnids were immobilized at the two highest test concentrations.
Microscopic observations of daphnids exposed to an average exposure concentration of 0.28 mg/L at 24 hours of exposure revealed no test item attached to their bodies.
The responses recorded in this test allowed for reliable determination of an EC50.

Experimental conditions
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 was maintained at 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Results with reference substance (positive control):

The actual responses in this reference test with K2Cr2O7 were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was within the expected range of 0.30 to 1.0 mg/L. Hence. the sensitivity of the tested batch of Daphnia magna was comparable to the sensitivity of batches previously tested at Charles River Den Bosch.
The 24h-EC50 was 0.55 mg/L with a 95% confidence interval ranging from 0.50 to 0.62 mg/L.
The 48h-EC50 was 0.39 mg/L with a 95% confidence interval ranging from 0.33 to 0.44 mg/L. The historical ranges for the 48h-EC50 lie between 0.28 and 0.90 mg/L. The observed 48h-EC50 corresponds with this range.

Reported statistics and error estimates:

None specified

Number of introduced daphnids and incidence of immobility in the combined limit/range-finding test

Time (h)	Replicate	Hepteen Base®; %SS at 100 mg/L
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5
	D	5
	Total introduced	20	10	10	10
24	A	0	0	5	5 (5)
	B	0	0	5 (1)	5 (5)
	C	0			5 (5)
	D	0			5 (5)
	Total immobilised	0	0	10	20
	Effect %	0	0	100	100
48	A	0	0	5	5^1
	B	0	1	5	5^1
	C	0			5^1
	D	0			5^1
	Total immobilised	0	1	10	100
	Effect %	0	10	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.

¹A light floating layer was observed in the test solution

 

 

Average exposure concentration versus nominal concentration in the final test

Hepteen Base® % SS at 100 mg/L	Measured concentrations (mg/L)		Average exposure (mg/L)
	t=0h	t=48h
0.46	0.071	0.037	0.051
1.0	0.14	0.081	0.11
2.2	0.37	0.21	0.28
4.6	0.67	0.39	0.51
10	1.4	0.90	1.1

 

 

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

Time (h)	Replicate	Hepteen Base®; Average exposure concentration (mg/L)
		Control	0.051	0.11	0.28	0.51	1.1
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	2	5	4
	B	0	0	0	0	5	5 (1)
	C	0	0	0	4	4	5
	D	0	0	0	4	4	5 (1)
	Total immobilised	0	0	0	10	18	19
	Effect %	0	0	0	50	90	95
48	A	0	0	1	5	5	5
	B	0	0	0	5	5	5
	C	0	0	1	5	5	5
	D	0	0	2	5	5	5
	Total immobilised	0	0	4	20	20	20
	Effect %	0	0	20	100	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.

 

pH and oxygen concentrations (mg/L) during the final test

Hepteen Base® Average exposure concentration (mg/L)	Start (t=0 h)		End (t=48 h)
	pH	O2	pH	O2
Control	8.0	9.0	8.0	9.4
0.051	8.0	8.9	8.1	9.4
0.11	8.0	8.9	8.0	9.3
0.28	8.0	8.9	8.0	9.4
0.51	8.0	8.9	8.0	9.4
1.1	8.0	8.9	8.0	9.4

 

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 was 0.15 mg/L based on average exposure concentrations (95% confidence interval between 0.18 and 0.19 mg/L).

Executive summary:

Acute Toxicity Study in Daphnia magna with Hepteen Base® (static).

Acute toxicity study using Hepteen Base® as conduced in daphnia magna in accordance with OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the CouncilRegulation (EC) No 440/2008,Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000.

The batch of Hepteen Base® tested was a clear amber liquid with a purity of 99.7% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring, after which the resulting mixture was allowed to stabilize for approximately 2 hours. Subsequently, the Saturated Solution (SS) was siphoned off through glass wool 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.

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 0.46, 1.0, 2.2, 4.6 and 10% of a SS prepared at a loading rate of 100 mg/L.The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Analysis of the samples taken at the start (t=0) of the final test showed measured concentrations of 0.071, 0.14, 0.37, 0.67 and 1.4 mg/L in solutions containing 0.46, 1.0, 2.2, 4.6 and 10% of a SS prepared at a loading rate of 100 mg/L, respectively. During the exposure period the measured concentration had decreased to 0.037, 0.081, 0.21, 0.39 and 0.90 mg/L, respectively. Based on these results, the average exposure concentrations were calculated.The range tested based on average measured concentrations was 0.051, 0.11, 0.28, 0.51 and 1.1 mg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The 48h-EC50 was 0.15 mg/L based on average exposure concentrations (95% confidence interval between 0.18 and 0.19 mg/L).

Description of key information

Acute Toxicity Study in Daphnia magna with Hepteen Base® (static).

Under the conditions of the study the test material met the criteria for classifying as Aquatic Acute 1, H400 according to Regulation (EC) 1272/2008

 

The 48h-EC50 was 0.15 mg/L based on average exposure concentrations (95% confidence interval between 0.18 and 0.19 mg/L).

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.15 mg/L

Additional information

Acute Toxicity Study in Daphnia magna with Hepteen Base® (static).

Acute toxicity study using Hepteen based was conduced in daphnia magna in accordance with OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No 440/2008,Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000.

The batch of Hepteen Base® tested was a clear amber liquid with a purity of 99.7% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.