4-benzyloxy-2-nitro-beta-pyrrolidinostyrene

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 to 13 april 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD guidance document no. 23, GUIDANCE DOCUMENT ON AQUATIC TOXICITY TESTING OF DIFFICULT SUBSTANCES AND MIXTURES, adopted 14. Dec. 2000

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pyrrolidine, 1-[2-[2-nitro-4-(phenylmethoxy)phenyl]ethenyl]-

Cas Number:

99474-22-3

Molecular formula:

C19H20N2O3

IUPAC Name:

Pyrrolidine, 1-[2-[2-nitro-4-(phenylmethoxy)phenyl]ethenyl]-

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae

The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

The algal medium corresponds to the OECD TG 201 medium.

Limit test:

no

Total exposure duration:

72 h

Test conditions

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 119.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 94.42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 97.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 81.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Any other information on results incl. tables

Findings

The cell numbers were determined with an electronic particle counter, the details are given in chapter 16, Annex 2: Detailed Data. The means and standard deviations of the cell numbers of the blank control and the treatments are presented in the following table:

Nominal Concentration in mg/L	Parameter		CellNumber/mL
			0 h		24 h		48 h		72 h
Blank control		Mean		2420		11153		56403		318640
Blank control		SD		0		1042		5668		28078
1		Mean		2420		10127		60200		296233
1		SD		0		771		3999		28223
3.2		Mean		2420		9533		40673		213680
3.2		SD		0		1756		10372		30790
10		Mean		2420		11487		60693		328980
10		SD		0		945		3231		2003
32		Mean		2420		11073		67180		349833
32		SD		0		775		9598		21496
100		Mean		2420		8293		27313		134787
100		SD		0		704		3110		15085

SD = Standard Deviation

Light intensity

	0 h	24 h	48 h	72 h
Lightintensity[Lux]	5000	5000	5000	5000

pH values

Nominal Concentrationin mg/L	0 h	72 h
Blank control	7.6	7.9
1	7.6	8.6
3.2	7.6	8.4
10	7.6	8.2
32	7.7	8.8
100	7.7	8.9

Analytical determination

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbonanalyser. Therefore IC and TC was measured for one replicate per treatment.

Because of the poor solubility, only in the highest concentration the test item could be detected in small quantities. Since the other treatments are all within the same range as the blank control, however, the statistical evaluation is based on the nominal values.

The details are given in the following tables:

Nominal Concentration	MeasuredTC  t = 0 h	MeasuredTC  t = 72 h	MeasuredIC  t = 0 h	MeasuredIC  t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	7.42	6.59	7.09	5.86
1	7.59	6.24	7.13	5.43
3.2	7.57	6.71	7.23	6.11
10	7.83	6.24	7.25	5.26
32	8.13	5.78	7.17	4.63
100	9.25	8.39	7.09	6.65

LOQ (Limit of quantification) TC = 5.43 mg/L .

LOQ (Limit of quantification) IC = 2.03 mg/L.

Nominal Concentration	Measured DOC (TC-IC)  t = 0 h	Measured DOC (TC-IC)  t = 72 h	Measured DOC minus blank control t = 0 h	Measured DOC minus blank control t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	0.33	0.73	--	--
1	0.46	0.81	0.14	0.08
3.2	0.34	0.60	0.02	-0.14
10	0.58	0.98	0.25	0.25
32	0.96	1.14	0.63	0.41
100	2.17	1.74	1.84	1.01

 

Nominal Concentration Test Item	Calculated Concentration  Test Item  t = 0 h	Calculated Concentration  Test Item  t = 72 h	% of Nominal concentration t = 0 h	% of Nominal concentration t = 72 h
mg/L	mg/L	mg/L	%	%
Blank control	--	--	--	--
1	0.2	0.1	19	11
3.2	0.0	-0.2	1	-6
10	0.4	0.4	4	4
32	0.9	0.6	3	2
100	2.6	1.4	3	1

 

Growth rate, yield

Nominal Concentration in mg/L	Parameter	GrowthRate (0-72h) [day^-1]	Yield (0-72h) [Cell Concentration/mL]
Blank control	Mean	1.63	316220
	SD	0.03	28078
1	Mean	1.60	293813
	SD	0.03	28223
3.2	Mean	1.49	211260
	SD	0.05	30790
10	Mean	1.64	326560
	SD	0.00	2003
32	Mean	1.66	347413
	SD	0.02	21496
100	Mean	1.34	132367
	SD	0.04	15085

SD = Standard Deviation

 

Inhibition

Nominal concentration in mg/L	% Inhibition
	GrowthRate (0-72h)	Yield(0-72h)
Blank control	0	0
1	1.5	7.1
3.2	8.3	33.2
10	-0.7	-3.3
32	-2.0	-9.9
100	17.7	58.1

 

Parameter	Value	95% confidenceinterval
NOEC (GrowthRate) 72 h	32mg/L	--
NOEC (Yield) 72 h	32 mg/L	--
LOEC (GrowthRate) 72 h	100mg/L	--
LOEC (Yield) 72 h	100mg/L	--
72h ErC10	94.42 mg/L	n.d.
72h EyC10	81.89 mg/L	n.d.
72h ErC50	119.32 mg/L	n.d.
72h EyC50	97.87 mg/L	n.d.

n.d. not determinable due to mathematical reasons

Validity

Parameter	Validitycriteria	Observed value	Assessment
Increase factorbiomass	factor 16in72 h	132	Valid
Mean coefficient of variation of daily growth rates	max. 35%	8%	Valid
Coefficient of variation of average growth rate during the whole test period	max. 7%	2%	Valid

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A GLP-study has been performed to assess the toxicity of the test item againstDesmodesmussubspicatusaccording to OECD 201 resp. EU C.3. The study was performed using 5 concentrations ranging from 1 to 100 mg/L nominal concentration. Incubation time was 72 hours. Significant inhibition of algal growth was observed at the highest concentration. The following results for the test item were determined:
NOECr = 32 mg/L
ErC50 = 119.32 mg/L
ErC10 = 94.42 mg/L
NOECb = 32 mg/L
EbC50 = 97.87 mg/L
EbC10 = 81.89 mg/L

Executive summary:

A GLP-study has been performed to assess the toxicity of the test item againstDesmodesmussubspicatusaccording to OECD 201 resp. EU C.3. The study was performed using 5 concentrations ranging from 1 to 100 mg/L nominal concentration. Incubation time was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the highest concentration.

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbonanalyser. Because of the poor solubility, only in the highest concentration the test item could be detected in small quantities. Since the other treatments are all within the same range as the blank control, however, the statistical evaluation is based on the nominal values.

The slight decrease in the test item concentration at the end of the test may be due to presence of the test organism (adsorption or ingestion onto the algae cells). Since the algae cells have been removed for DOC measurement.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item were determined:

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	32 mg/L	100 mg/L	94.42 mg/L	119.32 mg/L
Yield	32 mg/L	100 mg/L	81.89 mg/L	97.87 mg/L