Formaldehyde, oligomeric reaction products with acetone and diphenylamine

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:

17 August 2015 to 07 October 2015

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:

OECD guidelines for Testing of Chemicals, guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.2. "Daphnia Sp. Acute Immobilisation Test".

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Formaldehyde, oligomeric reaction products with acetone and diphenylamineAppearance: Dark brown flakesBatch: IC5B04P006Purity/Composition: 100% Unknown or Variable compositions, Complex reaction products and Biological materials (UVCB)Test substance storage: At room temperatureStable under storage conditions until: 26 February 2019 (expiry date)Purity/composition correction factor: No correction factor requiredTest substance handling: No specific handling conditions requiredChemical name (IUPAC), synonym or trade name: Formaldehyde, oligomeric reaction products with acetone and diphenylamine (BXA)CAS Number: 9003-80-9Molecular structure: UVCBMolecular formula: UVCBMolecular weight: UVCB

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from the limit concentration and the control according to the schedule below. In addition, filter containing undissolved residue was kept for possible analysis. Frequency: at t=0 h and t=48 hVolume: 2.0 ml from the approximate centre of the test vesselsStorage: Samples were stored in a freezer until analysis and transported on dry ice to the analytical site.At the end of the exposure period, the replicates were pooled at each concentration before sampling.Additionally, reserve samples of 2.0 ml were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

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.Water Accommodated Fractions (WAFs) were prepared at individual loading rates ranging from 1.0 to 100 mg/L. A two-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):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical 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 selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.BreedingStart 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 weeksRenewal of the cultures: After 7 days of cultivation half of the medium twice a week.Temperature of medium: 18-22°CFeeding: Daily, a suspension of fresh water algae.Medium: M7, as prescribed by Dr. Elendt-Schneider

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period specified in the study report

Hardness:

180 mg/L expressed as CaCO3

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

pH:

pH: 6.0-8.5, not varying by more than 1.5 units;

Dissolved oxygen:

oxygen: >3 mg/L at the end of the test).

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

The actual concentration measured at the start of the test was 2.0 mg/L. The measured concentration was at the level of 98% of nominal at the end of the test (i.e. 1.9 mg/L). This concentration is considered the maximum solubility in medium. The effect parameters were expressed in terms of measured concentrations.

Details on test conditions:

Test duration: 48 hoursTest type: StaticTest vessels: 100 ml, all-glassMedium: Adjusted ISO mediumNumber of daphnids: 20 per groupLoading: 5 per vessel containing 80 ml of test solutionLight: 16 hours photoperiod dailyFeeding: No feedingAeration: No aeration of the test solutions.Introduction of daphnids: Within 11 minutes after preparation of the test solutions.Measurements and recordingsImmobility (including mortality): At 24 hours and at 48 hours.pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control.Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: effect concentration exceed the maximum soluble concentration

Details on results:

Range-finding testNo immobility was observed at any of the test concentrations during the test period. Therefore, the expected EC50 was above loading rate of 100 mg/L.All test conditions were maintained within the limits prescribed by the protocol.Limit testMeasured concentrationsSamples taken from the limit concentration were analysed. The actual concentration measured at the start of the test was 2.0 mg/L. The measured concentration was at the level of 98% of nominal at the end of the test (i.e. 1.9 mg/L). This concentration is considered the maximum solubility in medium. The effect parameters were expressed in terms of measured concentrations.ImmobilityThe responses recorded in this test allowed for reliable determination of an EC50. As expected no effects were observed in the control and the limit concentration.Experimental conditionsThese test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, 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 varied between 20 and 21°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

Results with reference substance (positive control):

The study procedures described in this report were based on the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004 and the ISO International Standard 6341.The reference test was carried out to check the sensitivity of the test system as used by WIL Research Europe. Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l and to a control. Twenty daphnids were exposed per concentration.The reference substance, potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K44879664) was obtained from Merck, Darmstadt, Germany.The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/l. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at WIL Research Europe.The 24h-EC50 was estimated to correspond with 0.80 mg/l.The 48h-EC50 was estimated to correspond with 0.39 mg/l.

Reported statistics and error estimates:

Not specified

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

Time (h)	Replicate	Formaldehyde, oligomeric reaction products with acetone and diphenylamine; Loading rate (mg/L)
		Control	1.0	10	100
0	A B	5 5	5 5	5 5	5 5
	Total introduced	10	10	10	20
24	A B	0 0	0 0	0 0	0 0 (2)
	Total immobilised Effect %	0 0	0 0	0 0	0 0
48	A B	0 0	0 0	0 0 (1)	0 0
	Total immobilised Effect %	0 0	0 0	0 0	0 0

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

 

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

Time (h)	Replicate	Formaldehyde, oligomeric reaction products with acetone and diphenylamine; Loading rate (mg/L)
		Control	100
0	A B C D	5 5 5 5	5 5 5 5
	Total introduced	20	20
24	A B C D	0 0 0 0	0 0 0 0
	Total immobilised Effect %	0 0	0 0
48	A B C D	0 0 0 0	0 0 0 0
	Total immobilised Effect %	0 0	0 0

 

Effect parameters

Parameter	Formaldehyde, oligomeric reaction products with acetone and diphenylamine Loading rate (mg/L)
24h-EC50 48h-EC50	>2.0 >2.0

 

pH and oxygen concentrations during the final test

Formaldehyde, oligomeric reaction products with acetone and diphenylamine Measured concentration (mg/L)	Start (t=0 h)		End (t=48 h)
	pH	O2	pH	O2
Control 2.0	7.9 7.9	9.5 9.3	8.1 8.0	9.1 9.1

 

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate:

Concentration K2Cr2O7 (mg/l)	Number Exposed	% immobile		Expected response (%) After 48 hours^1
		24h	48h	Minimal	Maximal
Control 0.10 0.18 0.32 0.56 1.0 1.8	20 20 20 20 20 20 20	0 5 0 0 15 65 100	0 5 0 25 80 100 100	0 0 0 0 0 40 100	10^2 10 10 30 100 100 100

¹Based on historical data of the previous years (n>60)

²A maximum response of the 10% does not invalidate the results if the test

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 48h-EC50 exceeded the maximum soluble concentration, i.e. 2.0 mg/L.

Executive summary:

Acute Toxicity Study in Daphnia magna with Formaldehyde, oligomeric reaction products with acetone and diphenylamine.

 

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 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. 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.

 

A limit test was performed based on the results of a preceding range-finding test. Water Accommodated Fraction (WAF) was prepared at loading rate of 100 mg/L and used as the limit concentration.

 

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and the limit concentration. 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.

 

Samples taken from the limit concentration were analysed. The actual concentration was 2.0 mg/L at the start of the test and remained constant during the exposure (98% of nominal). This concentration is considered maximum soluble in medium. Therefore, the effect parameters were expressed in terms of measured concentrations.

 

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

 

The 48h-EC50 exceeded the maximum soluble concentration, i.e. 2.0 mg/L.

Description of key information

The key value was determined in a GLP accredited laboratory study in accordance OECD guideline No. 202 and EU Method C2.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2 mg/L

Additional information

An acute toxicity study in Daphnia magna was conducted with formaldehyde, oligomeric reaction products with acetone and diphenylamine. The substance was not completely soluble in test medium at the loading rates initially prepared. Before start of the preparation test substance was ground.

 

A limit test was performed based on the results of a preceding range-finding test. Water Accommodated Fraction (WAF) was prepared at loading rate of 100 mg/L and used as the limit concentration. Samples taken from the limit concentration were analysed. The actual concentration was 2.0 mg/L at the start of the test and remained constant during the exposure (98% of nominal). This concentration is considered maximum soluble in medium. Therefore, the effect parameters were expressed in terms of measured concentrations. The 48h-EC50 exceeded the maximum soluble concentration, i.e. 2.0 mg/L.