Phenol, 2,4-dinitro-, sulfurized, thiosulfonated

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20. - 27. March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

23rd March, 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009, Annex VI, C.26: „Lemna sp. Growth Inhibition Test“. Dated 24 August 2009

Version / remarks:

24 August 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Expiration date: 30 November 2021

Analytical monitoring:

yes

Details on sampling:

For preparation of test solutions a stock solution was first prepared by dissolving an amount of 0.1301 g test item in 1301 mL dilution water (20X AAP medium), resulting a stock solution of 100.0 mg/L nominal concentration. This stock solution was placed into an ultrasonic bath for 15 minutes. The test solutions of the chosen test concentrations were prepared by appropriate dilution of this stock solution. The test solutions were freshly prepared in the testing laboratory just before introduction of the test plants.

Vehicle:

no

Details on test solutions:

The test item solutions are prepared by mechanical dispersion of appropriate amount of test item in 20X AAP medium in order to obtain the concentration of 100 mg product/L and will be placed into the ultrasonic bath for approx. 15 minutes. The further test solutions (50.0, 25.0, 12.5 and 6.25 mg/L) will be prepared by appropriate dilution of this stock solution. Untreated control will be run parallel in the test. The test solution will be freshly prepared in the testing laboratory just before introduction of plants.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name (Strain): Lemna gibba (G3)
- Source: Friedrich Schiller Universität, Institut für Allgemeine Botanik und Pflanzenphysiologie,Jena, Germany
- Preculture: 7 - 10 days (7 days in this study) before testing, sufficient colonies are transferred from the stock culture aseptically into fresh sterile medium and cultured under the conditions of the test prior to beginning the test.
- Initial frond number: The initial frond number in the test cultures was 11. The number of colonies and fronds was identical in each test vessel.
- Replicates and controls: The test included three replicates at test concentrations and six replicates for the control group.

Test type:

static

Water media type:

other: 20X APP medium

Limit test:

yes

Total exposure duration:

7 d

Remarks on exposure duration:

7-day exposure in treated testing solutions; 5 concentrations with three replicates and the control group with six replicates. Assessment of growth and morphological changes on days 3, 5 and 7.

Test temperature:

24 ± 2 °C

pH:

The pH was checked at the start and at the end of the test in each concentration and the control. The pH of the control medium did not increase by more than 1.5 units during the test.

Nominal and measured concentrations:

Based on the results of the preliminary range-finding test five test concentrations in a geometric series with a separation factor of 2.0 and one control were used in the main test. The following nominal concentrations were tested: 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L.
The measured test item concentrations did not deviate more than 20 % from the nominal during the experiment therefore the biological results and endpoints based on the nominal concentrations.

Details on test conditions:

Test type: Static test. Based on the results obtained during analytical method validation (Study number: 805-100-2718) the test item is stable for the duration of 7 days in 20X AAP medium.
Temperature: The cultures were maintained at a temperature in the range of 24 ± 2 ºC which was checked at the beginning of the study and every 24 hours (in a surrogate flask filled with water in the climate chamber). In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber during the experimental period.
pH: The pH was checked at the start and at the end of the test in each concentration and the control. The pH of the control medium did not increase by more than 1.5 units during the test.
Light intensity: The test vessels were placed randomly on a tray, illuminated continuously at a light intensity between 6500-10000 lux using fluorescent light tubes (with a spectral range of 400-700 nm). The light intensity was checked and recorded at the start of the test at the position occupied by test containers. The differences in light intensity between the measurement points (i.e. position of fronds) did not exceed ± 15 % and therefore provided equal conditions for each test culture.
Test units: All-glass beakers (total capacity of 400 mL) were used and were covered by glass petri dishes. The volume of the test liquid in the vessels was 160 mL. Each test unit was uniquely identified with study code, test item concentration (and control) and replicate.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol is tested at least twice a year

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

Analytical Results
Concentrations of the test item were analytically determined at the start and at the end of the test.The measured concentrations did not deviate more than 20 % from the nominal and was stable throughout the study.

Biological Results
All biological results are based on the nominal concentrations.
The frond number in each test vessel was determined at the start and at the 3rd, 5th, 7th days.

Growth Rate
The average specific growth rate (based on frond number) was not statistically significantly different (growth inhibition) from the control group at treated concentrations (Dunnett t-Test; 1-sided smaller, α=0.05). Accordingly the NOEC related to growth rate (based on frond number) was determined to be 100 mg/L and the LOEC was determined to be >100 mg/L. The average specific growth rate (based on dry weight) was statistically significantly different from the control group at the highest concentration of 100 mg/L (Dunnett t-Test, 1-sided smaller, (α=0.05)). Accordingly the NOEC related to growth rate (based on dry weight) was determined to be 50.0 mg/L and the LOEC was determined to be 100.0 mg/L. The 168-h ErfnC50 was determined to be >100 mg/L (directly from the raw data). The 168-h ErdwC50 was determined to be > 100 mg/L (directly from the raw data).

Yield
The yield was calculated based on frond numbers and dry weights for each treatment and each parallel. Yield (0-7d) was not statistically significantly different (growth inhibition) from the control group in the treated concentrations based on frond number (Dunnett t-Test, 1-sided smaller, (α=0.05)). Accordingly the NOEC related to yield (based on frond number) was determined to be 100 mg/L and the LOEC was determined to be >100 mg/L. Yield (0-7d) was statistically significantly different from the control group in the highest concentration of 100 mg/L based on dry weight (Dunnett t-Test, 1-sided smaller (α=0.05)). Accordingly the NOEC related to yield (based on dry weight) was determined to be 50.0 mg/L and the LOEC was determined to be 100.0 mg/L. The 168-h EyfnC50 was determined to be >100 mg/L (directly from the raw data). The 168-h EydwC50 was determined to be >100 mg/L (directly from the raw data).

Results with reference substance (positive control):

For the evaluation of the quality of the Lemna gibba cultures and the experimental conditions, 3,5-dichlorophenol is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study with reference item 3,5-dichlorophenol was: 08 – 15 September 2017.
Endpoints of this study were: EyfnC50 (7 day, yield based on frond numbers): 3.980 mg/L, ErfnC50 (7 day, growth rate based on frond numbers): 6.053 mg/L, EydwC50 (7 day, yield based on dry weight): 2.906 mg/L, ErdwC50 (7 day, growth rate based on dry weight): 6.206 mg/L.

Reported statistics and error estimates:

For the determination of the LOEC and NOEC, Dunnett t-Test (1-sided smaller, α = 0.05) was used by SPSS PC+ software. Probit analysis was performed for determination of the EC10 values (based on dry weight) with 95 % confidence limits.
EC20 and EC50 values (based on dry weight) were given directly from the raw data.
EC10, EC20 and EC50 values (based on frond number) were given directly from the raw data.

Summary of the biological endpoints

Endpoints for response variables (0-7 d)		Concentration (mg/L) based on nominal concentrations
Growth Rate (based on frond number)	ErfnC10	>100
		not calculated
	ErfnC20	>100
		not calculated
	ErfnC50	>100
		not calculated
	NOEC	100
	LOEC	>100
Growth Rate (based ondry weight)	ErdwC10	71.69
	95 % conf. limits	44.077-194.842
	ErdwC20	>100
	95 % conf. limits	not determined
	ErdwC50	>100
	95 % conf. limits	not determined
	NOEC	50
	LOEC	100

Not calculated: Probit analysis has not been performed, since NOEC equals highest test concentration
Not determined: Probit analysis has been performed and did not yield results

 

Summary of the biological endpoints

Endpoints for response variables (0-7 d)		Concentration (mg/L) based on nominal concentrations
Yield (based on frond number)	EyfnC10	>100
		not calculated
	EyfnC20	>100
		not calculated
	EyfnC50	>100
		not calculated
	NOEC	100
	LOEC	>100
Yield (based on dry weight)	EydwC10	20.13
	95 % conf. limits	0.000-52.442
	EydwC20	>100
	95 % conf. limits	not determined
	EydwC50	>100
	95 % conf. limits	not determined
	NOEC	50
	LOEC	100

Not calculated: Probit analysis has not been performed, since NOEC equals highest test concentration
Not determined: Probit analysis has been performed and did not yield results

Solubilised Sulphur Black 1 concentrations measured during the study

Concentration, mg/L
Nominal	Start	End
	Mean of the measured concentrations (mg/L) with the 95% confidence intervals (n=3)		Percentage of the start concentration
100	112 ± 2.1	111 ± 4.0	99
50	57.8 ± 2.88	57.7 ± 5.4	100
25	28.0 ± 0.4	28.7 ± 0.7	103
12.5	13.9 ± 0.4	14.8 ± 0.5	107
6.25	6.92 ± 0.31	7.21 ± 1.02	104
Control	< LOD		-

Growth rates (µ) and percentage inhibition ofµbased on frond number
(Dunnett t-Test; 1-sided smaller,a= 0.05)

Concentration[mg/L]	Growth rate (r) and % inhibition of r
Nominal	0–168 h (based on frond number)
	r	% Ir
Control	0.330	-
6.25	0.351	-6.27
12.5	0.355	-7.59
25.0	0.375	-13.54
50.0	0.360	-8.98
100.0	0.359	-8.62

Remark: negative inhibition means growth stimulation

Growth rates (µ) and percentage inhibition ofµbased on dry weight

Concentration[mg/L]	Growth rate (r) and % inhibition of r
Nominal	0–168 h (based on dry weight)
	r	% Ir
Control	0.35149	-
6.25	0.36298	-3.27
12.5	0.33493	4.71
25.0	0.33526	4.62
50.0	0.32681	7.02
100.0	0.30808*	12.35

*: statistically significantly different compared to the control values (Dunnett t-Test;1-sided smaller, a = 0.05)

Remark: negative inhibition means growth stimulation

 Yield (y) and percentage inhibition of yield based on frond number (Dunnett t-Test;a= 0.05, smaller 1-sided)

Concentration[mg/L]	Yield and % inhibition of yield
Nominal	0–168 h (based on frond number)
	y	% Iy
Control	100.17	-
6.25	118.00	-17.80
12.5	121.67	-21.46
25.0	141.00	-40.77
50.0	126.33	-26.12
100.0	125.33	-25.12

Remark: negative inhibition means growth stimulation

Yield (y) and percentage inhibition of yield based on dry weight

Concentration[mg/L]	Yield and % inhibition of yield
Nominal	0–168 h (based on dry weight)
	y	% Iy
Control	0.00618	-
6.25	0.00671	-8.61
12.5	0.00542	12.27
25.0	0.00544	12.00
50.0	0.00509	17.56
100.0	0.00444*	28.08

*: statistically significantly different compared to the control values (Dunnett t-Test;a= 0.05, 1-sided smaller)

Remark: negative inhibition means growth stimulation

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, based on the results of the study the ErC50 and NOEC (growth rate based on frond number) was determined to be >100 mg/L and 100 mg/L, respectively, for test item Solubilised Sulphur Black 1.

Executive summary:

The influence of the test item Solubilised Sulphur Black 1 on the growth of the freshwater aquatic plant Lemna gibba (duckweed) was investigated in a 7-day static test, based on the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test” (2006) and the Commission Regulation (EC) No. 761/2009, C.26. Exponentially growing cultures of Lemna gibba were exposed for a period of 7 days in a static test system to the test item at nominal concentrations of 6.25, 12.5, 25.0, 50.0, and 100.0 mg/L. Based on the results the ErC50 and NOEC (growth rate based on frond number)for the test item was determined to be >100 mg/L and 100 mg/L, respectively.

Description of key information

In a Growth Inhibition Test according to OECD guideline 221, an ErC50 and NOEC value (based on growth rate and frond number) of >100 mg/L and 100 mg/L were determined, respectively.

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

50 mg/L

Additional information

The influence of the test item Solubilised Sulphur Black 1 on the growth of the freshwater aquatic plant Lemna gibba (duckweed) was investigated in a 7-day static test, based on the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test” (2006) and the Commission Regulation (EC) No. 761/2009, C.26. Exponentially growing cultures of Lemna gibba were exposed for a period of 7 days in a static test system to the test item at nominal concentrations of 6.25, 12.5, 25.0, 50.0, and 100.0 mg/L. Based on the results the ErC50 and NOEC (growth rate based on frond number) for the test item was determined to be >100 mg/L and 100 mg/L, respectively.