4-(hydroxymethyl)-4-methyl-1-phenylpyrazolidin-3-one

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Remarks:

lemna

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2017 to 28 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-(hydroxymethyl)-4-methyl-1-phenylpyrazolidin-3-one

EC Number:

235-920-3

EC Name:

4-(hydroxymethyl)-4-methyl-1-phenylpyrazolidin-3-one

Cas Number:

13047-13-7

Molecular formula:

C11H14N2O2

IUPAC Name:

4-(hydroxymethyl)-4-methyl-1-phenylpyrazolidin-3-one

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Concentrations: all concentrations on day 0 and 6 (fresh) and on day 1 and 7 (24h-old) --> 2.0 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: anaylsed freshly
- sample preparation:diluted in a 1:1 (v:v) ratio with 1% L-ascorbic acid in Imodified Steinberg medium and analyzed ( the substance is known to be stabel in 0.5% L-ascorbic acid in water)

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of test solutions started with the highest concentration of 100 mg/L with magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. At the end of the preparation procedure, all test solutions were clear and colorless besides the highest concentration that was observed to be slightly yellow.
Due to the light sensitive nature of the test item, preparation was performed under dimmed light and the glassware was wrapped in aluminum foil.

Test organisms

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: lemna minor (duckweed)
- Source (laboratory, culture collection): in-house culture (started at the Friedrich-Schiller-Universität Jena)
- Method of cultivation: exposed to light in a climate room at a temperature of 22-26°C.

ACCLIMATION: 7 days before the start of the test, pre-cultures were started with 2 or 3 plants each with a maximum of 3 fronds. The total number of fronds increased by a factor of ≥ 7 in 7 days with a doubling time of ≤ 2.5 days. The culture consisted of young, rapidly growing colonies with bright green fronds that varied in size without visible lesions, chlorosis, gibbosity or necrosis. The pre-culture was maintained under the same conditions as used in the test.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Hardness:

no data

Test temperature:

23-25°C

pH:

fresh 5.2-5.7
old 4.8-6.1 (increase over duration of the study)

Dissolved oxygen:

no data

Nominal and measured concentrations:

nominal: 0, 1.0, 3.2, 10, 32 and 100 mg/L
measured (TWA): 0, 0.80, 2.6, 7.5, 24 and 88 mg/L (see table)

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: yes (23 - 25°C)
- Test vessel: 250 ml, all-glass, containing 150 ml of test solution
- Aeration: no
- Agitation: yes swirled
- Renewal rate of test solution: density
- No. of fronds per colony: 3 plants, with a total of 9 fronds
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (modified Steinberg medium according to the guideline)

TEST MEDIUM / WATER PARAMETERS: Modified Steinberg medium; according to ISO 20079, formulated using Milli-RO water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: TLD-lamps Intensity at the start: 92-102 µE.m-2.s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting at renewals
- Determination of biomass: fresh weight on day 0 and 7
- Determination of frond area: no data

RANGE-FINDING STUDY
- Test concentrations: 0, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: expected EC50 for the inhibition of yield (no of fronds) > 100 mg/L, expected EC50 for inhibition of yield (biomass) 10-100 mg/L
- Analytics: The actual concentrations measured in the freshly prepared solutions were at the level of 19-96% of nominal at the start of the test and 29-93% of nominal in the last renewal period. The actual concentrations decreased to 30-49% of initially measured at the end of the first and 22-28% of initially measured in the last renewal period. This finding is not in line with the findings of the method development study and therefore in the main study samples were analysed freshly (immediately after taking without storage).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 88 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: growth rate

Effect concentrations 2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

frond number

Remarks on result:

other: growth rate and yield

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

55 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: growth rate

Effect concentrations 4

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

< 0.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: growth rate and yield

Details on results:

see tables
Plants exposed to the two highest concentrations were observed to be smaller than the control plants from day 4 of exposure. From day 6 of exposure plants exposed to the three highest concentrations were observed to be smaller and have shorter roots than the control plants.

Reported statistics and error estimates:

Determination of the NOEC/LOEC and calculation of the ECx (x=10, 20, 50)
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.
The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Growth rate based on frond numbers

 

Period (day)	Replicate	CH01610, Average exposure concentration (mg/L)
		Control	0.80	2.6	7.5	24	88
0-7	1	0.356	0.379	0.34	0.324	0.319	0.264
	2	0.378	0.347	0.371	0.338	0.276	0.256
	3	0.371	0.383	0.36	0.308	0.293	0.264
	4	0.348
	5	0.338
	6	0.34
Mean:		0.355	0.37	0.357	0.324	0.296	0.261
Std.Dev.:		0.0165	0.0198	0.016	0.0148	0.0218	0.0045
n:		6	3	3	3	3	3
CV:		4.7	5.3	4.5	4.6	7.4	1.7
		0.362	0.358	0.317	0.335	0.339	0.277

 

The doubling time of frond number in the control was 2.0 days, corresponding to a
12-fold increase in seven days and an average specific growth rate of 0.355 d^-1.

Individual Biomass (g)

Time	CH01610, Average exposure concentration (mg/L)
	Control	0.80	2.6	7.5	24	88
0 h	0.0170	0.0170	0.0170	0.0170	0.0170	0.0170
	0.0170	0.0170	0.0170	0.0170	0.0170	0.0170
	0.0170	0.0170	0.0170	0.0170	0.0170	0.0170
	0.0170
	0.0170
	0.0170
n:	6	3	3	3	3	3
Mean:	0.0170	0.0	0.0	0.0	0.0	0.0
7 d	0.1977	0.1176	0.1254	0.0881	0.0778	0.0558
	0.2381	0.1430	0.1238	0.0965	0.0588	0.0487
	0.1972	0.1642	0.1398	0.0794	0.0663	0.0538
	0.1677
	0.1651
	0.1784
n:	6	3	3	3	3	3
Mean:	0.1907	0.1416	0.1297	0.088	0.0676	0.0528
Std.Dev.:	0.0271	0.0233	0.0088	0.0086	0.0096	0.0037
CV:	14	16.5	6.8	9.7	14.1	6.9

     
Individual Growth Rates (Biomass)

Time	CH01610, Average exposure concentration (mg/L)
	Control	0.80	2.6	7.5	24	88
7 d	0.351	0.276	0.285	0.235	0.217	0.17
	0.377	0.304	0.284	0.248	0.177	0.15
	0.35	0.324	0.301	0.22	0.194	0.165
	0.327
	0.325
	0.336
Mean:	0.344	0.302	0.29	0.234	0.196	0.162
Std.Dev.:	0.0195	0.024	0.0095	0.0139	0.0201	0.0101
n:	6	3	3	3	3	3
CV:	5.7	7.9	3.3	5.9	10.2	6.2

      
Individual Yield (Biomass)

Time	CH01610, Average exposure concentration (mg/L)
	Control	0.80	2.6	7.5	24	88
7 d	0.2	0.1	0.1	0.1	0.1	0
	0.2	0.1	0.1	0.1	0	0
	0.2	0.1	0.1	0.1	0	0
	0.2
	0.1
	0.2
Mean:	0.2	0.1	0.1	0.1	0.1	0
Std.Dev.:	0.03	0.02	0.01	0.01	0.01	0
n:	6	3	3	3	3	3
CV:	15.6	18.7	7.8	12	18.9	10.2

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC50 and NOEC for growth rate based on biomass are 55 and < 0.8 mg/L
The EC50 and NOEC for growth rate based on frond number are > 88 and 0.8 mg/L

Executive summary:

Six replicates of Lemna minor colonies were exposed to a control, whereas three replicates per group were exposed to substance concentrations of 1.0, 3.2, 10, 32 and 100 mg/L. The total exposure period was 7 days and test solutions were renewed daily. The initial plant density per replicate was 3 with a total frond number of 9 per replicate. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the first and the last renewal period.

The actual concentrations measured in the freshly prepared solutions and ages soutions showed a time weighed average exposure concentrations were 0.80, 2.6, 7.5, 24 and 88 mg/L at nominally1.0, 3.2, 10, 32 and 100 mg/L, respectively.

A dose-related response were observed for all relevant endpoints.

The effect parameters (mg/L) obtained in this study are summarise in tables below.

		Parameter (mg/L)	NOEC	LOEC	EC10	EC20	EC50
Frond numbers	Growth rate	Value	2.6	7.5	11	43	>88
		lower 95%-cl			5.1	29	n.d.
		upper 95%-cl			18	63	n.d.
	Yield	Value	2.6	7.5	2.9	8.4	64
		lower 95%-cl			0.69	3.5	40
		upper 95%-cl			6.0	14	140
Biomass	Growth rate	Value	<0.80	0.80	0.55^1	2.7	55
		lower 95%-cl			0.30	1.8	43
		upper 95%-cl			0.87	3.6	76
	Yield	Value	<0.80	0.80	0.11^1	0.41^1	5.3
		lower 95%-cl			0.057	0.26	4.3
		upper 95%-cl			0.18	0.59	6.4

n.d. – not determined,¹– indicative value obtained by extrapolation of the dose-response curve

In conclusion, under the conditions of the present study with Lemna minor, CH01610 reduced growth rate and inhibited the yield of this fresh water plant species significantly at 7.5 mg/L (LOEC) when calculated based on frond number and at 0.80 mg/L when calculated based on biomass. The NOEC for growth rate reduction and yield inhibition was 2.6 mg/L (fronds) and <0.80 mg/L (biomass).