Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 2015 to 01 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD guidelines for Testing of Chemicals, guideline No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006; Annex 5 corrected 28 July 2011.

Deviations:

yes

Remarks:

In the range-finding test, cell densities were determined only at the end of the test. Evaluation: This provided sufficient information for the final test.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C3: “Algal Inhibition Test”; Amended by EC No. 761/2009 of 23 July 2009, Publication No. L220.

Deviations:

yes

Remarks:

In the range-finding test, cell densities were determined only at the end of the test. Evaluation: This provided sufficient information for the final test.

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 all groups according to the schedule below. In addition, filter containing undissolved residue was kept for possible analysis.Frequency: at t=0 h, t=24 h and t=72 hVolume: 2.0 mlStorage: 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 with algae were pooled at each concentration before sampling.Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.Additionally, reserve samples of 2.0 ml were taken from all test solutions for possible analysis. If not already 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:

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.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.After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1Source: In-house laboratory culture.Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford,Mass., USA)Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1E4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

Not specified

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C.Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

pH:

The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit).

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Samples taken from the limit concentration were analysed. The actual concentration at the start of the test was 2.1 mg/L. The measured concentration decreased to 1.2 mg/L. Based on these results, the Time Weight Average (TWA) concentrations were calculated.The concentrations measured in the limit concentration incubated without algae showed similar results. This indicates, that the observed decrease of concentration was not caused by the growing algal biomass.

Details on test conditions:

Range-finding testA 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 limit test with the following exceptions:-Exponentially growing algal cultures were exposed to a WAFs prepared at 1.0, 10 and 100 mg/L-Three replicates were tested per concentration and three replicates in the control group.-One extra test vessel per concentration without algae was used as background for the determination of the algal cell density.-pH was only measured in the control and the highest test concentration.-Cell densities were determined only at the end of the test.-No sampling for determination of actual test concentrations was performed.Limit testTest concentrationsFormaldehyde, oligomeric reaction products with acetone and diphenylamine: WAF prepared at 100 mg/LControls: Test medium without test substance or other additivesReplicates: 6 replicates of the control and 6 replicates of the limit concentration. 1 replicate of each group for sampling purposes at 24 hour of exposure. 2 replicates of the limit concentration without algae.Test procedures and conditionsTest duration: 72 hoursTest type: StaticTest vessels: 100 ml, all-glass, containing 50 ml of test solutionMedium: M2Cell density: An initial cell density of 1 x 104 cells/ml.Illumination: Continuously using TLD-lamps with a light intensity within the range of 79 to 85 μE.m-2.s-1.Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.MeasurementspH: At the beginning and at the end of the test.The pH of the control solutions should preferably not deviate by more than 1.5 units during the test.Temperature of medium: Continuously in a temperature control vessel.Appearance of the cells: At the end of the final test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae.Recording of cell densitiesAt the beginning of the test, cells were counted using a microscope and a counting chamber.Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Mean cell densities, inhibition of growth rate and inhibition of yieldBased on the results the expected EC50 for growth rate and yield inhibition exceeded the loading rate of 100 mg/L.All test conditions were maintained within the limits prescribed by the protocol.Limit testMeasured test substance concentrationsSamples taken from the limit concentration were analysed. The actual concentration at the start of the test was 2.1 mg/L. The measured concentration decreased to 1.2 mg/L. Based on these results, the Time Weight Average (TWA) concentrations were calculated.The concentrations measured in the limit concentration incubated without algae showed similar results. This indicates, that the observed decrease of concentration was not caused by the growing algal biomass.Inhibition of growth rate and inhibition of yieldNo biologically significant differences were recorded between the values for growth rate at any of the test concentrations when compared to the control group. Statistically significant inhibition of yield was found at the limit concentrations, however the observed effect was considered biologically insignificant (<10%).The yield was inhibited by 13% at the limit concentration. This was considered statistically significant.Therefore, the NOEC for yield inhibition could not be determined.Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.Experimental conditionsThe pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

Results with reference substance (positive control):

Not specified

Reported statistics and error estimates:

Not specified

Mean cell densities (x10⁴cells/ml) during the range-finding test

Time (h)	Formaldehyde, oligomeric reaction products with acetone and diphenylamine; Loading rate (mg/L)
	Control	1.0	10	100
0 72	1.0 229.7	1.0 236.2	1.0 228.8	1.0 201.7

 

Percentage inhibition of growth rate during the range-finding test

Test substance^1Loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control 1.0 10 100	1.812 1.821 1.811 1.767	0.0206 0.0149 0.0119 0.0493	3 3 3 3	-0.5 0.1 2.5

^1.Formaldehyde, oligomeric reaction products with acetone and diphenylamine

 

Percentage inhibition of yield during the range-finding test

Test substance^1Loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control 1.0 10 100	228.7 235.2 227.9 200.8	14.03 10.58 8.07 29.01	3 3 3 3	-2.8 0.4 12.2

^1.Formaldehyde, oligomeric reaction products with acetone and diphenylamine

 

Measured concentrations versus nominal concentrations

Test substance^1Loading rate (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72h
100 100 WA	2.14 2.16	1.96 1.92	1.23 0.718	1.7 1.5

^1.Formaldehyde, oligomeric reaction products with acetone and diphenylamine

WA – without algae

 

Percentage inhibition of growth rate (total test period) during the final test

Test substance^1TWA concentration (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control 1.7	1.890 1.843	0.0122 0.0180	6 6	2.5^*#

^1.Formaldehyde, oligomeric reaction products with acetone and diphenylamine

^*- effect was statistically significant

^#- effect biologically insignificant (<10%)

 

Percentage inhibition of growth rate at different time intervals during the final test

Test substance^1TWA concentration (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72 h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6 3	2.487 2.528	0.0 -1.6	1.776 1.638	0.0 7.8	1.406 1.353	0.0 3.1

^1.Formaldehyde, oligomeric reaction products with acetone and diphenylamine

 

Percentage inhibition of yield during the final test

Test substance^1TWA concentration (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control 1.7	289.0 251.2	10.64 13.54	6 6	13.1^*

^1.Formaldehyde, oligomeric reaction products with acetone and diphenylamine

^*- effect was statistically significant

 

Effect parameters

Parameter (mg/L)	NOEC	EC10	EC20	EC50
Growth rate Yield	≥1.7 <1.7	>1.7 <1.7	>1.7 >1.7	>1.7 >1.7

 

pH levels recorded during the final test

Test substance^1TWA concentration (mg/L)	pH
	t=0h	t=72h
Control 0.17	8.1 8.1	8.1 8.1

^1.Formaldehyde, oligomeric reaction products with acetone and diphenylamine

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition was recorded at the limit concentration of Formaldehyde, oligomeric reaction products with acetone and diphenylamine tested.The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded a TWA concentration of 1.7 mg/L. This concentration is being considered the maximum soluble in medium.The 72h-NOEC for growth rate inhibition was higher or equal 1.7 mg/L.The 72h-NOEC for yield inhibition could not be determined.Due to the very low solubility of Formaldehyde, oligomeric reaction products with acetone and diphenylamine in water, concentration levels that might be toxic for algae could not be reached.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with Formaldehyde, oligomeric reaction products with acetone and diphenylamine.

 

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/ 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). Test substance 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.

 

Six exponentially growing algal cultures per group, were exposed to an untreated control and to the limit concentration. Initial cell density was 104 cells/ml. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

 

Samples taken from the limit concentration were analysed. The actual concentration at the start of the test was 2.1 mg/L. The measured concentration decreased to 1.2 mg/L. Based on these results, the Time Weight average concentration was calculated to correspond to 1.7 mg/L and was considered maximum soluble in the medium.

 

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

 

Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition was recorded at the limit concentration of Formaldehyde, oligomeric reaction products with acetone and diphenylamine tested.

 

The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded a TWA concentration of 1.7 mg/L. This concentration is being considered the maximum soluble in medium.

 

The 72h-NOEC for growth rate inhibition was higher or equal 1.7 mg/L.

The 72h-NOEC for yield inhibition could not be determined.

 

Due to the very low solubility of Formaldehyde, oligomeric reaction products with acetone and diphenylamine in water, concentration levels that might be toxic for algae could not be reached.

Description of key information

The key value was determined in a GLP accredited laboratory study performed in accordance with OECD guideline No. 201 and EU Method Part C.3.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.7 mg/L

EC10 or NOEC for freshwater algae:

1.7 mg/L

Additional information

A fresh water algal growth inhibition test was conducted in Pseudokirchneriella subcapitata with formaldehyde, oligomeric reaction products with acetone and diphenylamine.

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 at the start of the test was 2.1 mg/L. The measured concentration decreased to 1.2 mg/L. Based on these results, the Time Weight average concentration was calculated to correspond to 1.7 mg/L and was considered maximum soluble in the medium.

 

Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of growth rate or inhibition was recorded at the limit concentration of formaldehyde, oligomeric reaction products with acetone and diphenylamine tested. The EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) was beyond the range tested, i.e. exceeded a TWA concentration of 1.7 mg/L. This concentration is being considered the maximum soluble in medium. The 72h-NOEC for growth rate inhibition was higher or equal 1.7 mg/L. The 72h-NOEC for yield inhibition could not be determined.

 

Due to the very low solubility of formaldehyde, oligomeric reaction products with acetone and diphenylamine in water, concentration levels that might be toxic for algae could not be reached.