Heptanal, oligomeric reaction products with aniline

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 October 2016 to 25 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

yes

Remarks:

See "Any other infomration on materials and methods incl. tables" for more details

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

2008

Deviations:

yes

Remarks:

See "Any other infomration on materials and methods incl. tables" for more details

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
During the final test, single samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t = 0h and t = 96h
Volume: 2.0 mL
Storage: Not applicable, samples were analysed on the day of 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 99.70% 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. 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):

Cyprinus carpio

Details on test organisms:

Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length: Range-finding test: 4.1 ± 0.4 cm / Final test: 2.4 ± 0.2 cm
Mean weight Range-finding test: 0.94 ± 0.26 g / Final test: 0.51 ± 0.4 g
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 51

Holding
Quarantine/Acclimatisation: At least 12 days after delivery
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, erkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 2 11.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 4 6.7 mg/L
KCl 4.2 mg/L
Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality parameters: Were kept within the optimum limits for the respective fish species.
Feeding: Daily with pelleted fish food (Essence (300-500 um), Coppens International bv, Helmond, The Netherlands)
Acclimatisation: Following a 48-hour settling-in period the fish were allowed to acclimatise to the test medium without test item for at least seven days.
Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not specified

Hardness:

180 mg CaCO3

Test temperature:

20-24°C

pH:

pH: 6.0-8.5

Dissolved oxygen:

> 60%

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Measured concentration: 0.42, 0.94, 2.0, 4.0 and 8.9 mg/L in 4.6, 10, 22, 46 and 100% of the SS prepared at 100 mg/L

Details on test conditions:

Test procedure and conditions
Test duration: 96 hours
Test type: Static
Test vessels: 8 litres, all-glass, containing 7 litres of test solution.
Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
Number of fish: 7 per concentration
Loading: 0.51 g fish/Litre, i.e. 7 fish per 7 litres of test medium
Illumination: 16 hours photoperiod daily
Aeration: Aeration was introduced after 72 hours of exposure.
Feeding: No feeding from 24 hours prior to the test and during the total test period
Introduction of fish: Within 21 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 0.5 units and 0.5°C.
Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

Range-finding test
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three fish per concentration were exposed to a range of 1.0 to 100% of a SS prepared at 100 mg/L increasing by a factor of 10.
• Dissolved oxygen concentrations, pH and temperature were only measured in the lowest and the highest test concentration with surviving fish.

Final test-Test concentrations
Hepteen Base®: Solutions containing 4.6, 10, 22, 46 and 100% of a SS prepared at 100 mg/L.
Controls: Test medium without test item or other additives.

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%-confidence interval (mg/L) 4.0-8.9

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%-confidence interval (mg/L) 2.0-8.9

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

5.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%-confidence interval (mg/L) 2.0-8.9

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%-confidence interval (mg/L) 3.1-11

Details on results:

Range-finding test
No mortality or clinical effects were observed at the two lowest test concentrations during the test period. At the highest concentration, all fish died within 24 hours of exposure. The expected LC50 was between 10 and 100% SS.
Samples taken from all concentrations were analysed. The initial concentrations were 0.085, 0.90 and 9.1 mg/L in 1.0, 10 and 100% SS, respectively. All concentrations remained stable during the test period (94-104% of initial at the end of the test.
All test conditions were maintained within the limits prescribed by the study plan.

Final test
Measured concentrations
The results of analysis of the samples taken during the final test are described in Table 4 of the appended Analytical Report.
Samples taken from all test concentrations were analysed. At the start of the test, the actual concentrations were 0.42, 0.94, 2.0, 4.0 and 8.9 mg/L in 4.6, 10, 22, 46 and 100% of the SS prepared at 100 mg/L, respectively. These concentrations remained stable throughout the test duration, i.e. were at 99-109% of initial at the end of the test.
Based on these results, the initially measured concentrations were used to determine the effect concentrations.

Mortality and other effects
No mortality or clinical effects were observed at the three lowest test concentrations during the test period. At 4.0 mg/L, damage to the fins of the surviving fish was observed and mortality increased with increasing exposure duration. At the highest test concentration, all fish had died after 24 hours of exposure.
The responses recorded in this test allowed for reliable determination of a 96h-LC50. The responses recorded were in agreement with the results of the range-finding test.

Experimental conditions
Note that aeration was introduced after 72 hours of exposure, as the oxygen concentration in one test solution had dropped below the optimum level for testing with carp, i.e. below 5.0 mg/L.
All test conditions remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).

Results with reference substance (positive control):

During the test the pH, oxygen concentration and the temperature of the medium were within the optimal ranges for fish.

Under the conditions of the present test all carp exposed to a PCP concentration of 0.46 mg/l died within 24 hours. One fish died at 0.22 mg/l and no mortality occurred at 0.10 mg/l. The 96h-LC50 for carp exposed to PCP was 0.24 mg/l. This effect was already reached within 48 hours of exposure.

The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/l based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.

Sublethal observations / clinical signs:

Incidence of mortality and total mortality during the range-finding test

Hepteen Base® % SS prep. at 100 mg/L	Initial number of fish	Cumulative mortality				Total Mortality (%)
		24h	48h	72h	96h
1.0	3	0	0	0	0	0
10	3	0	0	0	0	0
100	3	3	3	3	3	100

 

Incidence of mortality and total mortality during the final test

Hepteen Base® Concentration (mg/L)	Initial Number of fish	Cumulativemortality				Total Mortality (%)
		24h	48h	72h	96h
Control	7	0	0	0	0	0
0.42	7	0	0	0	0	0
0.94	7	0	0	0	0	0
2.0	7	0	0	0	0	0
4.0	7	0	1	2	3	43
8.9	7	7	7	7	7	100

 

Clinical effects observed during the final test

Hepteen Base® Concentration (mg/L)	Time of recording (hours)	Specification of effects	Relative number
Control, 0.42, 0.94, 2.0 4.0	0-96 24 48 72 96	No effects Visible damage of fins Visible damage of fins Visible damage of fins^1 Visible damage of fins^1	7/7 7/7 6/6 5/5 4/4

¹Precipitation of undissolved material was observed in the test solution.

Effect parameters

Parameter	Hepteen Base® Concentration (mg/L)	95%-confidence interval (mg/L)
24h-LC50	6.0	4.0-8.9
48h-LC50	5.6	2.0-8.9
72h-LC50	5.1	2.0-8.9
96h-LC50	4.3	3.1-11

 

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

Hepteen Base® Concentration (mg/L)	Day 0		Day 1		Day 2		Day 3		Day 4
	pH^1	O2	pH	O2	pH	O2	pH	O2	pH	O2
Control	7.7	9.6	7.6	7.6	7.6	6.5	7.6	6.3	7.8	7.5
0.42	7.7	9.4	7.5	7.0	7.4	6.7	7.5	6.1	7.8	8.1
0.94	7.7	9.4	7.5	6.7	7.4	6.4	7.4	5.3	7.8	7.1
2.0	7.7	9.1	7.5	7.0	7.4	6.8	7.4	5.6	7.8	6.5
4.0	7.8	9.1	7.5	7.4	7.4	6.3	7.3	3.8	7.8	8.0
8.9	7.9	8.6	7.6	7.9	-	-	-	-	-	-

¹          pH of culture medium was: 7.7

Temperatures (°C) measured during the final test

Hepteen Base® Concentration (mg/L)	Day 0^1	Day 1	Day 2	Day 3	Day 4
Control	20.9	21.6	21.6	21.8	21.3
0.42	20.9	21.7	21.8	22.0	21.8
0.94	21.0	21.7	21.7	21.9	21.7
2.0	21.1	21.8	21.9	22.1	21.6
4.0	21.1	21.8	21.8	22.0	21.8
8.9	21.5	21.8	-	-	-

¹  Temperature of culture medium was: 21.4°C.

 

Validity criteria fulfilled:

yes

Conclusions:

The 96h-LC50 was 4.3 mg/L based on measured exposure concentrations (95% confidence interval between 3.1 and 11 mg/L).

Executive summary:

A 96-Hour acute toxicity study using Hepteen Base in Carp was conducted in accordance with OECD guideline No. 203, 1992.In addition, the procedures were designed to meet the test methods of theCouncilRegulation (EC) No 440/2008,Part C.1, 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 99.70% and not completely soluble in test medium at the loading rate initially prepared.

 

Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirringafter which the resulting mixture was allowed to stabilize for 1 hour. Subsequently, the Saturated Solution (SS) was siphoned off. 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 range-finding test. Seven carp per test group were exposed to an untreated control and to Hepteen Base® test groups containing 4.6, 10, 22, 46 and 100% of a SS prepared at a loading rate of 100 mg/L. Samples for analytical confirmation of actual exposure concentrations were taken at the start of the test and after 96 hours of exposure.

 

Samples taken from all test concentrations were analysed. At the start of the test, the actual concentrations were 0.42, 0.94, 2.0, 4.0 and 8.9 mg/L in 4.6, 10, 22, 46 and 100% of the SS prepared at 100 mg/L, respectively. These concentrations remained stable throughout the test duration, i.e. were at 99-109% of initial at the end of the test. Based on these results, the initially measured concentrations were used to determine the effect concentrations.

 

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

The 96h-LC50 was 4.3 mg/L based on measured exposure concentrations (95% confidence interval between 3.1 and 11 mg/L).

 

Description of key information

96-Hour Acute Toxicity Study in Carp with Hepteen Base® (Static)

The 96h-LC₅₀was 4.3 mg/L based on measured exposure concentrations (95% confidence interval between 3.1 and 11 mg/L).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.3 mg/L

Additional information

96-Hour Acute Toxicity Study in Carp with Hepteen Base® (Static)

Key value was determined in a GLP accredited laboratory in accordance with OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No 440/2008, Part C.1, 2008 and the OECD series on testing and assessment number 23, 2000.