Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

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:

2017-11-17 to 2017-11-25

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:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009, Annex VI, C.26

Version / remarks:

August 24, 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Six replicate samples were analysed from the test solutions at the start and at the end of the renewal periods. Six replicate samples were analysed from the control as well. The samples were concentrated 5-fold before the measurement.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: As the test item is poorly soluble in deionized water as well in the test medium, preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). 0.1500 g, 0.1502 g and 0.1505 g of the test item was suspended in 1500 mL of the dilution water (20X AAP Medium; see 5.4) in the first, second and third renewal periods, respectively. The loading rate of the test solutions was 100 mg test item/L at the start of each renewal period. The test solution was handled by ultrasonic bath for 10 minutes thereafter stirred rigorously for a period of 24 hours to achieve an equilibrated test concentration. The test solution was then filtrated through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. The test solution was freshly prepared in the testing laboratory just before introduction of the test organisms and at start of each renewal period.

Test organisms (species):

Lemna gibba

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed
- Strain: G3
- Source (laboratory, culture collection): In-house culture kept under axenic conditions; preculture was prepared at least 7 days before the tests; healthy colonies with 3-4 fronds. Originally, the culture was obtained from 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.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Post exposure observation period:

Not performed.

Hardness:

Not specified

Test temperature:

24 +/- 2 °C

pH:

Control: 7.77 - 8.73
Test solution: 8.51 - 8.71

Dissolved oxygen:

Not specified

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 0.023 mg/L (mean)

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: Yes
- Test vessel: All-glass beakers (total capacity of 400 mL) were used and were covered by glass petri dishes, with total capacity of 400 mL were used. 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.
- Material, size, headspace, fill volume: Glass, total capacity = 400 mL, fill volume = 160 mL, - Type: Closed
- Type of cover: covered by glass petri dishes
- Aeration: No
- Agitation: No
- Renewal rate of test solution: Renewed twice during the test (on days 3 and 5).
- No. of fronds per colony: 11
- No. of vessels per concentration: 6
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes (20 X AAP Medium)

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Illuminated continuously
- Light intensity and quality: 6500-10000 lux (with a spectral range of 400-700 nm)

EFFECT PARAMETERS MEASURED
- Determination of frond number: Manual counting
- Determination of biomass: Measured as frond counts

RANGE-FINDING STUDY (non-GLP)
- Test concentrations: 100 mg (nominal)
- Results used to determine the conditions for the definitive study: Yes. Based on the results obtained during analytical method validation the test item is not regarded to be stable for the duration of 7 days in 20X AAP medium. Therefore the test and control solution were renewed twice during the test (on days 3 and 5).

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 0.023 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.023 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.023 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.023 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 0.023 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Details on results:

- Any visual signs of phytotoxicity: No
- Decrease in frond size: No
- Necrosis / chlorosis: No
- Sinking of fronds: No
- Any stimulation of growth found in any treatment: No

Results with reference substance (positive control):

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:

A limit test was performed; therefore, statistical analysis to determine the EC values was not necessary. For the determination of the LOEC and NOEC, the calculated mean growth rate and yield at the calculated test concentration were tested on significant differences to the control value using analysis of variance (ANOVA) and Independent Sample T-test by SPSS PC+ software program. The data were checked for homogeneity of variance by Levene’s test.

Table 1: Growth rates (µ) and percentage inhibition of µ based on frond number

Concentration (mg/L)		Growth rate (µ) and % inhibition of µ
Nominal	Measured	0–168 h(based on frond number)
		µ	% Iµ
Control	-	0.364	-
100 (WAF)	0.023	0.362	0.67

 Table 2: Growth rates (µ) and percentage inhibition ofµbased on dry weight

Concentration (mg/L)		Growth rate (µ) and % inhibition of µ
Nominal	Measured	0–168 h(based on dry weight)
		µ	% Iµ
Control	-	0.36643	-
100 (WAF)	0.023	0.37149	-1.38*

*: negative value indicates increase in comparison to the control

Table 3: Yield (y) and percentage inhibition of yield based on frond number

Concentration (mg/L)		Yield (y) and % inhibition of yield
Nominal	Measured	0–168 h(based on frond number)
		y	% Iy
Control	-	130.33	-
100 (WAF)	0.023	127.67	2.05

Table 4:Yield (y) and percentage inhibition of yield based on dry weight

Concentration (mg/L)		Yield (y) and % inhibition of yield
Nominal	Measured	0–168 h(based on dry weight)
		y	% Iy
Control	-	0.00691	-
100 (WAF)	0.023	0.00713	-3.21^*

*: negative value indicates increase in comparison to the control.

Table 5:Symptoms, changes ofLemna gibbaplants observed during the test

Concentration (mg/L)		3^rdday of the experiment		5^thday of the experiment		7^thday of the experiment
Nominal	Measured	Symptoms	Degree of change	Symptoms	Degree of change	Symptoms	Degree of change
Control	-	–	–	–	–	–	–
100 (WAF)	0.023	–	–	–	–	–	–

Legend:

"–":the plants were healthy, there was not any symptom observed

Validity criteria fulfilled:

yes

Conclusions:

In a Lemna sp. Growth Inhibition Test according to OECD Guideline 221 and Commission Regulation (EC) No 761/2009, Annex VI, C.26, the 7-d EC50 and the NOEC of the test item was > 0.023 mg/L (arithmetric mean measured; > 100 mg/L in nominal).

Executive summary:

The toxicity of the test item to aquatic plants was assessed in a GLP-compliant Lemna sp.Growth Inhibition Test according to OECD Guideline 221 and Commission Regulation (EC) No 761/2009, Annex VI, C.26. Exponentially-growing cultures of Lemna gibba were exposed to the nominal concentration of 100 mg/L (highest concentration to be tested according to OECD 221) of the test item. Due to the low solubility of the test item, test solutions (six replicates) were prepared by utilizing the water accommodated fraction (WAF) approach method (according to OECD Series on Testing and Assessment No. 23). In addition a negative control (20 X AAP Medium; six replicates) and a positive control with the reference substance 3,5-dichlorophenol was performed. 400 mL Petri dishes-covered glass beakers were filled with 160 mL testing solutions. Two Lemna gibba colonies with four, and one colony with three fronds were added to each vessel. Visual assessment of growth and morphological changes was conducted on days 3, 5 and 7. As the test item is not stable for the duration of 7 days in 20 X AAP medium, the test and control solution were renewed twice during the test (on days 3 and 5). The test item concentration was measured by UV-Vis to be 0.023 mg/L (arithmetric mean). In result, the 7-d EC50 and NOEC of the test item was determined to be > 0.023 mg/L (arithmetric mean measured). In conclusion, the test item had no toxic effect at aquatic saturation (i.e. limit test concentration) on Lemna gibba; the overall LOEC is higher than the nominal concentration of 100 mg/L (solubility level) of the test item in the test medium, which corresponds to the mean measured concentration of 0.023 mg/L, based on water accomodated fraction. The validity criterion of the test guidelines was within the acceptable limit and therefore the study can be considered as valid.

Description of key information

In a Lemna sp.Growth Inhibition Test according to OECD Guideline 221 and Commission Regulation (EC) No 761/2009, Annex VI, C.26 with a WAF, the 7-d EC50 and the NOEC were higher than the solubility level of the test item in the test medium, which corresponds to a mean measured concentration of 0.023 mg/L (EC50: >100 mg/L nominal).

Additional information

The toxicity of the test item to aquatic plants was assessed in a GLP-compliant Lemna sp.Growth Inhibition Test according to OECD Guideline 221 and Commission Regulation (EC) No 761/2009, Annex VI, C.26. Exponentially-growing cultures of Lemna gibba were exposed to the nominal concentration of 100 mg/L (highest concentration to be tested according to OECD 221) of the test item. Due to the low solubility of the test item, test solutions (six replicates) were prepared by utilizing the water accommodated fraction (WAF) approach method (according to OECD Series on Testing and Assessment No. 23). In addition a negative control (20 X AAP Medium; six replicates) and a positive control with the reference substance 3,5-dichlorophenol was performed. 400 mL Petri dishes-covered glass beakers were filled with 160 mL testing solutions. Two Lemna gibba colonies with four, and one colony with three fronds were added to each vessel. Visual assessment of growth and morphological changes was conducted on days 3, 5 and 7. As the test item is not stable for the duration of 7 days in 20 X AAP medium, the test and control solution were renewed twice during the test (on days 3 and 5). The test item concentration was measured by UV-Vis to be 0.023 mg/L (arithmetric mean). In result, the 7-d EC50 and NOEC of the test item was determined to be > 0.023 mg/L (arithmetric mean measured). The doubling time of frond number in the control was 1.90 days (less than 2.5 days). Thus, the validity criterion of the test guidelines was within the acceptable limit and therefore the study can be considered as valid.