Formaldehyde, oligomeric reaction products with acetone and diphenylamine

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:

18 November 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:

OECD guidelines for Testing of Chemicals, guideline No. 203: "Fish Acute Toxicity Test", Adopted 17 July, 1992.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

Council Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.1. "Acute Toxicity for Fish".

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Specific details on test material used for the study:

Purity/composition correction factor: No correction factor required
Test substance handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: ormaldehyde, oligomeric reaction products with acetone and diphenylamine (BXA)
CAS Number: 9003-80-9
Molecular structure: UVCB

Analytical monitoring:

yes

Details on sampling:

During the final test samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h, t=24 h and t=96 h
Volume: 2.0 mL
Storage: Not applicable, samples were analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

The batch of Formaldehyde, oligomeric reaction products with acetone and diphenylamine tested consisted of dark brown flakes. The composition of the substance was unknown (UVCB). The substance was not completely soluble in test medium at the loading rates initially prepared. Before start of the preparation test substance was ground. No correction was made for the purity/composition of the item.

Water Accommodated Fractions (WAFs) were prepared at individual loading rates ranging from 1.0 to 100 mg/L. A three-day period of magnetic stirring was applied to accelerate dissolution of the test substance in medium. Thereafter, the obtained mixtures were filtered through a 0.45 µm membrane filter (RC 55, Whatman) and used as test concentrations.

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: 3.3 ± 0.2 cm
Mean weight: 1.1 ± 0.18 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: 20

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, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O; 211.5 mg/L
MgSO4.7H2O; 88.8 mg/L
NaHCO3; 46.7 mg/L
KCl; .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)
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:

No post exposure observation period specified in the study report.

Hardness:

180 mg CaCO3 per litre

Test temperature:

temperature 20-24°C

pH:

pH: 6.0-8.5

Dissolved oxygen:

oxygen > 60% of air saturation

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Test item: WAFs prepared at loading rates of 1.0, 10 and 100 mg/L
Controls: Test medium without test item or other additives

Details on test conditions:

Test procedure and conditions
Test duration: 96 hours
Test type: static
Test vessels: Control and the limit concentration: 20 litres, all-glass, containing 20 litres of test solution; Lower concentrations: 10 litres, all-glass, containing 8 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: Control and the limit concentration: 7 per group; Lower concentrations: 3 per group
Loading: Control and the limit concentration: 0.38 g fish/Litre, i.e. 7 fish per 20 litres of test medium; Lower concentrations: 0.41 g fish/Litre, i.e. 3 fish per 8 litres of test medium
Illumination: 16 hours photoperiod daily
Aeration: The test media were not aerated during the test.
Feeding: No feeding from 24 hours prior to the test and during the total test period
Introduction of fish: Within 33 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 unit 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.

Measurements and recordings
Mortality and other effects: At 4¼, 24, 48, 72 and 96 hours following the start of exposure. In addition, every morning from day 1 to observe for any dead or severely distressed fish.
Dissolved oxygen content: Daily in the control and the limit concentration, beginning at
pH and temperature: the start of the test (day 0).

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 616 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Measured concentrations
Concentrations of two components (m/z 170 and m/z 391.2) were determined in this study.. The results of m/z 391.2 appeared to be not reliable and therefore, the effect parameters were based only on the results obtained for m/z 170.
The actual concentrations measured at the start of the test increased from 23 µg/L at the lowest loading rate to 758 µg/L at the limit concentration. The measured concentrations were stable during the first 24 hours of exposure (87-90% of initial). At the end of the exposure the actual concentrations were at the level of 62-65% of initial at the two highest concentrations and at the lowest it decreased to 34% of initial.

Mortality and other effects
No mortality or clinical effects were observed during this test. The responses recorded in this test allowed for reliable determination of an LC50.
The test solutions were clear and colorless during the exposure. At 48 hour of exposure faeces were observed in the highest concentration

Experimental conditions
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.

Reported statistics and error estimates:

Not specified in the study report.

Sublethal observations / clinical signs:

Time Weight Average (TWA) concentration versus nominal concentration

Test item^1 Loading rate (mg/L)	Measured concentration (μg/L) at			TWA (μg/L)
	0 h	24 h	96 h
1.0	23.4	20.7	8.01	15
10	229	200	141	179
100	758	686	491	616

¹. Formaldehyde, oligomeric reaction products with acetone and diphenylamine

 

Incidence of mortality and total mortality during the final test

Test item^1 TWA conc. (μg/L)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		4 ¼ h	24 h	48 h	72 h	96 h
Control	7	0	0	0	0	0	0
15	3	0	0	0	0	0	0
179	3	0	0	0	0	0	0
616	7	0	0	0	0	0	0

¹. Formaldehyde, oligomeric reaction products with acetone and diphenylamine

 

Effect parameters

Parameter	Test item^1 TWA conc. (μg/L)
24, 48, 72 and 96 h – LC50	> 616

¹. Formaldehyde, oligomeric reaction products with acetone and diphenylamine

 

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

Test item^1 TWA conc. (μg/L)	Day 0		Day 1		Day 2		Day 3		Day 4
	pH^2	O2	pH	O2	pH	O2	pH	O2	pH	O2
Control	7.9	9.3	7.6	7.6	7.5	6.6	7.4	7.1	7.7	7.0
616	8.1	9.2	7.6	8.2	7.5	6.6	7.4	6.5	7.6	6.7

¹. Formaldehyde, oligomeric reaction products with acetone and diphenylamine

². pH of culture medium was: 8.0

 

Temperature (°C) measured during the final test

Test item^1 TWA conc. (μg/L)	Day 0^2	Day 1	Day 2	Day 3	Day 4
Control	21.2	21.5	21.5	21.7	21.7
616	20.8	21.5	21.6	21.8	21.5

¹. Formaldehyde, oligomeric reaction products with acetone and diphenylamine

². Temperature of culture medium was: 21.3°C

 

Incidence of mortality observed in the reference study:

Concentration PCP (mg/l) Nominal	Initial Number Of Fish	Cumulative number of dead fish recorded at various time points after start of exposure					Total Mortality (%)
		4 h	24 h	48 h	72 h	96 h
0.10	5	0	0	0	0	0	0
0.22	5	0	0	1	1	1	20
0.46	5	0	5	5	5	5	100

 

Validity criteria fulfilled:

yes

Conclusions:

The 96h-LC50 was beyond the range tested, i.e. exceeded the TWA concentration of 616 µg/L obtained in the WAF prepared at a loading rate of 100 mg/L and being considered maximum soluble in test medium.

Executive summary:

96-Hour Acute Toxicity Study in Carp with Formaldehyde, oligomeric reaction products with acetone and diphenylamine (static).

 

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.1, 2008 and the OECD series on testing and assessment number 23, 2000.

 

The batch of Formaldehyde, oligomeric reaction products with acetone and diphenylamine tested consisted of dark brown flakes. Before start of the preparation test substance was ground. The composition of the substance was unknown (UVCB). The substance was not completely soluble in test medium at the loading rates initially prepared.

 

A combined limit/range-finding test was performed. Water Accommodated Fractions (WAFs) were prepared at individual loading rates ranging of 1.0, 10 and 100 mg/L and used as test concentrations.

 

Seven fish per group were exposed to an untreated control and the limit concentration prepared at a loading rate of 100 mg/L (limit test). In addition, three fish per group were exposed to WAFs prepared at 1.0 and 10 mg/L in a combined range-finding test. The total exposure period was 96 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 hours and at the end of the test.

 

Concentrations of two components (m/z 170 and m/z 391.2) were determined in this study. The results of m/z 391.2 appeared to be not reliable and therefore, the effect parameters were based only on the results obtained for m/z 170.

 

The actual concentrations (based on m/z 170) measured at the start of the test increased from 23 µg/L at the lowest loading rate to 758 µg/L at the limit concentration. The measured concentrations were stable during the first 24 hours of exposure (87-90% of initial). At the end of the exposure the actual concentrations were at the level of 62-65% of initial at the two highest concentrations and at the lowest it decreased to 34% of initial.

 

Based on these results, the Time Weight Average (TWA) concentrations were calculated and correspond with 15, 179 and 616 µg/L at 1.0, 10 and 100 mg/L, respectively.

 

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

 

The 96h-LC50 was beyond the range tested, i.e. exceeded the TWA concentration of 616 µg/L obtained in the WAF prepared at a loading rate of 100 mg/L and being considered maximum soluble in test medium.

Description of key information

The key value was determined in a GLP accredited laboratory study using Carp in accordance with OECD Guideline No. 203 & EU Method C.1.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

616 µg/L

Additional information

A 96 -hour acute toxicity study was conducted in Carp with formaldehyde, oligomeric reaction products with acetone and diphenylamine (static). The substance was not completely soluble in test medium at the loading rates initially prepared. 

A combined limit/range-finding test was performed. Water Accommodated Fractions (WAFs) were prepared at individual loading rates ranging of 1.0, 10 and 100 mg/L and used as test concentrations.

 

Seven fish per group were exposed to an untreated control and the limit concentration prepared at a loading rate of 100 mg/L (limit test). In addition, three fish per group were exposed to WAFs prepared at 1.0 and 10 mg/L in a combined range-finding test. The total exposure period was 96 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 hours and at the end of the test.

 

Concentrations of two components (m/z 170 and m/z 391.2) were determined in this study. The results of m/z 391.2 appeared to be not reliable and therefore, the effect parameters were based only on the results obtained for m/z 170.

The actual concentrations (based on m/z 170) measured at the start of the test increased from 23 µg/L at the lowest loading rate to 758 µg/L at the limit concentration. The measured concentrations were stable during the first 24 hours of exposure (87-90% of initial). At the end of the exposure the actual concentrations were at the level of 62-65% of initial at the two highest concentrations and at the lowest it decreased to 34% of initial.

 

Based on these results, the Time Weight Average (TWA) concentrations were calculated and correspond with 15, 179 and 616 µg/L at 1.0, 10 and 100 mg/L, respectively. The 96h-LC50 was beyond the range tested, i.e. exceeded the TWA concentration of 616 µg/L obtained in the WAF prepared at a loading rate of 100 mg/L and being considered maximum soluble in test medium.