2-Propanone, reaction products with 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:

19 February 2018 to 22 February 2018

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 Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 28th July, 2011.

Deviations:

yes

Remarks:

See "Any other information" for details.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACh), Annex IV Part C, C.3 (published in the Official Journal of the European Union L 220 of 24 August 2009).

Deviations:

yes

Remarks:

See "Any other information" for details.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164, April 1996.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Based on the agreement with the sponsor and due to the fact that the Test Item is an UVCB the analytical measurement was performed by TOC analysis. Only the sum of the carbon content of the Test Item could be measured this way (and this did not change by the end of the test, independently from the stability), measurement was sufficient only at the beginning and at the end (additional vessel without algae) of the experiment to justify that the Test Item was present in the test solution, since more accurate data cannot be provided by TOC. Sample from the control was taken for analysis at the beginning and end of the experiment.

All samples were analysed directly after sampling.

Vehicle:

no

Details on test solutions:

Because the Test Item is a UVCB (Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials), a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. A saturated Test Item solution at 100 mg/L Test Item nominal loading rate (WAF) was prepared individually by dispersing/dissolving the needed amount of Test Item into the test medium (OECD medium) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30°C and then was equilibrated for about 24 hours at approximately 20°C. The non-dissolved test materials were removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solution. As only limit test was carried out, further dilution of this saturated solution was not performed.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of four days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

not specified

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Not specified.

Test temperature:

The temperature was 22.5°C measured in the flask and 21.7 and 23.2 °C measured within the climate chamber.

pH:

The range of the pH was 7.19 – 8.77 during the experiment.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

No toxicity was observed in the preliminary concentration range-finding test, therefore 100 mg/L Test Item nominal loading rate (WAF) and one control was used in the main test.

Details on test conditions:

TEST CONDITIONS
Temperature
Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was 22.5°C measured in the flask and 21.7 and 23.2 °C measured within the climate chamber.

pH
The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.19 – 8.77 during the experiment.

Light Intensity
The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7813 lux (equivalent to ~106 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

DESCRIPTION OF THE TEST PROCEDURE
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.
The test was performed with 6 replicates per test concentration and 6 replicates in the control group. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Preliminary Range Finding Tests
A concentration range-finding test was conducted to determine the approximate toxicity of the Test Item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the Test Item plus a control, for 72 hours. The test was performed with 2 replicates per each test concentration and 3 replicates in the control group.
During the formulation procedure the solutions were prepared individually.
At all applied test concentrations (including the control) the algal cells were normal, no morphological deviations were observed.

Test item concentrations in the Definitive Test
No toxicity was observed in the preliminary concentration range-finding test, therefore 100 mg/L Test Item nominal loading rate (WAF) and one control was used in the main test. The concentrations in the definitive test were based on the results of the preliminary range-finding test. The biological results are based on the nominal test item concentrations.

OBSERVATIONS
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in the test concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

other: growth rate, cell number & yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

other: growth rate, cell number & yield

Key result

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate WAF

Basis for effect:

other: growth rate, cell number & yield

Details on results:

VALIDITY
The cell density in the control cultures increased by the factor of 69.83 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 14.70 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 1.91 %.
All validity criteria were met; therefore, the study can be considered as valid.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
There were no morphological deviations of the algal cell during the study in any of the groups.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 17/367-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704):
04–07 December 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h average specific growth rate, yield and areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Loading Rate (NOELR) determined as 100 mg/L nominal loading rate WAF.

Results of the Preliminary Range-Finding Test

Nominal concentrations [mg/L nominal loading rates WAFs]	Untreated control	0.1	1	10	100
Average of cell number at 72 hours (x 10^4cell/mL)	71.7	71.0	70.5	69.0	68.5

 

Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Test group	Growth rate μ and % inhibition of μ
	0-24 h		0-48 h		0-72 h
	μ	%	μ	%	μ	%
Control	0.0569	0.0	0.0594	0.0	0.0589	0.0
100 mg/L nominal loading rate WAF	0.0533	6.2	0.0586	1.4	0.0582	1.2

 

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group	Area under the Growth Curves (A) and % inhibition of A
	0-24 h		0-48 h		0-72 h
	A	%	A	%	A	%
Control	36.0	0.0	268.0	0.0	1290.0	0.0
100 mg/L nominal loading rate WAF	32.0	11.1	252.0	6.0	1224.0	5.1

 

Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group	Yield (Y) and % inhibition of y (0-72 h)
	Y	%
Control	68.8	0.0
100 mg/L nominal loading rate WAF	65.3	5.1

 

pH-Values in the Test Media at the Start and End of the Test

Test group	pH-values
	Start	End
Control	7.20	8.50
		8.49
		8.58
		8.43
		8.57
		8.60
100 mg/L nominal loading rate WAF	7.19	8.77
		8.63
		8.56
		8.56
		8.51
		8.53

 

Temperature in the Climate Chamber and in the Test Media during the Test

Parameter		Exposure Time
		Day 0	Day 1	Day 2	Day 3
Temperature (°C) measured in flask		22.5	22.5	22.5	22.5
Temperature (°C) of the climate chamber	Min	21.7	21.9	22.0	22.0
	Max	23.0	23.2	23.2	23.2

 

Cell Number (x 10⁴cell/mL) determined in the Main Experiment

Test group		Number of cells
		0 h	24 h	48 h	72 h
Control		1	3	17	70
		1	4	18	79
		1	5	16	65
		1	5	17	64
		1	4	18	67
		1	3	18	74
	Mean	1.00	4.00	17.33	69.83
	SD	0.0	0.9	0.8	5.8
100 mg/L nominal loading rate WAF		1	5	16	70
		1	3	16	60
		1	4	18	64
		1	3	16	65
		1	3	16	66
		1	4	18	73
	Mean	1.00	3.67	16.67	66.33
	SD	0.0	0.8	1.0	4.6

 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the study, the Test Item had no toxic effect at saturation; the EL50 results and the LOELR are higher than the solubility level of the Test Item in the test medium.

The 72h EbL50 value: > 100 mg/L nominal loading rate WAF
The 72h ErL50 value: > 100 mg/L nominal loading rate WAF
The 72h EyL50 value: > 100 mg/L nominal loading rate WAF
The 72h NOELR: 100 mg/L nominal loading rate WAF
The 72h LOELR: > 100 mg/L nominal loading rate WAF

Executive summary:

The effect of the Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

 

As no toxicity was observed in the preliminary concentration range-finding test, therefore 100 mg/L Test Item nominal loading rate (WAF) and one control was used in the main test.

 

The concentration of the total carbon content was analytically determined at the start and at the end of the experiment. The biological results are based on the nominal concentration.

 

The test design included 6 replicates at test concentration and 6 replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a= 0.05) by TOXSTAT software.

 

The ErL50, EbL50 and EyL50 values of the Test Item were determined directly from the raw data.

 

The 72h EbL50 value:       > 100 mg/L nominal loading rate WAF

The 72h ErL50 value:       > 100 mg/L nominal loading rate WAF

The 72h EyL50 value:       > 100 mg/L nominal loading rate WAF

The 72h NOELR:              100 mg/L nominal loading rate WAF

The 72h LOELR:              > 100 mg/L nominal loading rate WAF

 

In conclusion, under the conditions of this study, the Test Item had no toxic effect at saturation; the EL50 results and the LOELR are higher than the solubility level of the Test Item in the test medium.

Description of key information

The 72h EbL50 value:       > 100 mg/L nominal loading rate WAF

The 72h ErL50 value:       > 100 mg/L nominal loading rate WAF

The 72h EyL50 value:       > 100 mg/L nominal loading rate WAF

The 72h NOELR:                   100 mg/L nominal loading rate WAF

The 72h LOELR:                    > 100 mg/L nominal loading rate WAF

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The effect of the Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

As no toxicity was observed in the preliminary concentration range-finding test, therefore 100 mg/L Test Item nominal loading rate (WAF) and one control was used in the main test.

The test design included 6 replicates at test concentration and 6 replicates for the untreated control.

 

The ErL50, EbL50 and EyL50 values of the Test Item were determined directly from the raw data.

The 72h EbL50 value:       > 100 mg/L nominal loading rate WAF

The 72h ErL50 value:       > 100 mg/L nominal loading rate WAF

The 72h EyL50 value:       > 100 mg/L nominal loading rate WAF

The 72h NOELR:                   100 mg/L nominal loading rate WAF

The 72h LOELR:                    > 100 mg/L nominal loading rate WAF

 

In conclusion, under the conditions of this study, the Test Item had no toxic effect at saturation; the EL50 results and the LOELR are higher than the solubility level of the Test Item in the test medium.

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