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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07. September 2020 - November 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]
EC Number:
225-935-3
EC Name:
Barium bis[2-chloro-5-[(2-hydroxy-1-naphthyl)azo]toluene-4-sulphonate]
Cas Number:
5160-02-1
Molecular formula:
C17H13ClN2O4S.1/2Ba
IUPAC Name:
barium(2+) bis(5-chloro-2-[(1E)-2-(2-hydroxynaphthalen-1-yl)diazen-1-yl]-4-methylbenzene-1-sulfonate)
Test material form:
solid: nanoform
Specific details on test material used for the study:
Name of test substance: Pigment Red 53:1
CAS No.: 5160-02-1
Homogeneity: Given
Physical state/Appearance: Solid / red
Density: 1.73 g/cm3

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Experiment 1 – Test with the complete dispersion:
Sampling of the complete dispersion Samples for concentration control analysis were taken from each freshly prepared solution (0h) without Lemna and at the end of each exposure interval (approx. 24 hours) from all test vessels (pooled replicates), as well as from the uninoculated (without Lemna) test vessel of each concentration group. For each concentration, two retained samples were taken at each time point. Retain samples were stored at the Laboratory ECO in a freezer (at -20 °C). Analysis of these samples were performed in case of equivocal analytical results with the original (primary) samples or after loss of/damage to original samples after agreement by the Study Director. Analysis is documented and justified in both, the raw data and report. Retain samples are described by the suffix “B” and “C” in the report. Following finalization of the report, all analytical samples, including retain samples, were discarded.

Experiment 2 – Test with the dissolved fraction:
Samples for concentration control analysis were taken from each freshly prepared solution (0h) without Lemna and at the end of each exposure interval (approx. 24 hours) from all test vessels (pooled replicates), as well as from the uninoculated (without Lemna) test vessel of each concentration group, according to the sampling scheme below. Only a subset of the primary samples was sent to the analytical laboratory for concentration control analyses, as indicated in the sampling scheme. For each concentration, two retained samples were taken at each time point. Retain samples will be stored at the Laboratory ECO in a freezer (at -20 °C). Analysis of these samples were performed in case of equivocal analytical results with the original (primary) samples or after loss of/damage to original samples after agreement by the Study Director. Analysis are documented and justified in both, the raw data and report. Retain samples are described by the suffix “B” and “C” in the report. Following finalization of the report, all analytical samples, including retain samples, were discarded. All samples were taken with a pipette and transferred into 4.0 mL amber glass sample vials.

Test solutions

Vehicle:
no
Details on test solutions:
Experiment 1 – Test with the complete dispersion:
For each concentration a 2L dispersion was prepared. The dispersion was prepared in 1L-batches only, i.e. to receive 2L of dispersion, two separate 1L batches were mixed. The 1L dispersions were prepared from sonicated test substance concentrates and made up with test medium. Each concentrate was prepared by weighing the respective amount of test substance into a rosette-cell (RZ 3, Bandelin, Germany) and adding 70 mL of purified water. Each concentrate was sonicated in an ultrasonic bath for approximately 30 minutes to ensure a thorough wetting of the test substance. Afterwards, each concentrate was sonicated by applying an energy of approx. 3 kJ/mL (corresponds in total to approx. 210 kJ) with a BANDELIN Sonopuls HD4400 unit equipped with a TS413- Sonotrode and set to an amplitude of 30%. Finally, each concentrate was transferred into a volumetric flask and the respective amount of test medium was added. The dispersions were stirred until use. The test medium was prepared under the conditions of the test substance dispersions, i.e. 70 mL of purified and sonicated water was transferred into a volumetric flask and filled up to 1L with test medium. The test medium was prepared in 1L-batches only and mixed to receive the final volume. The test medium was stirred until its use. Fresh test dispersions were prepared daily. Acceptable test solution pH is within the range ±0.5 of the control. If a test solution is outside of this range, it will be adjusted to control media pH (±0.2) with HCl or NaOH prior to the start of exposure. No pH adjustment of the test solutions was done prior or during the study. Following preparation, the test substance dispersions showed a redish clear coloration (1-32. mg/L) with a concentrationdependent increasing dispersion-like and colored appearance (10- 100 mg/L).

Experiment 2 – Test with the dissolved fraction:
The test substance is a mixture that is poorly soluble in water. Therefore, the test solutions were prepared following general guidance provided in OECD 23 to achieve a water accommodated fraction (WAF) of the test substance 4L of a WAF-solution was prepared by means of two 2Lpreparations. To receive at least 3L of WAF-supernatant, the gained separate supernatants were mixed. Each WAF-preparation was prepared by adding the respective test-substance amount to 2L of test medium in 2L glass bottles and stirring at ~70 rpm (to avoid dispersion formation) on a magnetic stirrer for approximately 48 hours. Afterwards, the preparation was left undisturbed for about additional 24 hours to facilitate the sedimentation of undissolved particles. Then the “sandwiched” supernatant between the top- and bottom layer of undissolved test substance was carefully obtained with a PVC (polyvinyl chloride) tube and filtered twice through a 0.2μm PES (polyethersulfone) filter. The gained dissolved-fraction solutions were inspected visually for the presence of any undissolved test substance by observing the scattering of a laser light through the test solution (Tyndall effect). Acceptable test solution pH is within the range ±0.5 of the control. If a test solution is outside of this range, it will be adjusted to control
media pH (±0.2) with HCl or NaOH prior to the start of exposure. No pH adjustment of the test solutions was done prior or during the study. Following preparation, the test solutions containing test substance showed an orange clear coloration. Tyndall effect was negative for all prepared test solutions. Fresh dissolved-fraction solutions were prepared daily.

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Strain: Lemna gibba Clone G3
- Source (laboratory, culture collection): The stock culture was obtained from BASF SE, Crop Protection Ecology and Environmental Analytics, Speyerer Strasse 2, 67117 Limburgerhof, Germany.
- Age of inoculum (at test initiation): An inoculum culture of Lemna gibba, 7 and 9 days old, (Experiment 1 and 2, respectively), at 24 °C ± 2°) is used to initiate the tests (study day 0).
- Method of cultivation: A culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared with sufficient colonies of Lemna transferred aseptically into fresh sterile 20X AAP media. The inoculum culture is incubated under test conditions for 9 days prior to test initiation.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d

Test conditions

Test temperature:
24 ±2°C
pH:
6.5 ± 0.2
Conductivity:
265 μS/cm (25 °C)
Nominal and measured concentrations:
EXPERIMENT 1 – Test with the complete dispersion:
0 (control), 1, 3.2, 10, 32, and 100 mg/L as nominal concentrations, based on test substance mass without correction for purity.
0 (control), 0.8, 2.6, 9.5, 26.1, and 69 mg/L as mean analytically measured (geometric mean) concentrations, considering biotic samples.

EXPERIMENT 2 – Test with the dissolved fraction:
0 (control), and 100 mg/L as nominal loading rate, based on test substance
mass without correction for purity.
0 (control), and 1.8 mg/L as mean analytically measured (geometric mean) concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Tall form glass beaker covered with petri dishes (nominal volume 400 mL, colorless with a nominal depth of 100 mm).
- Test volume: 300 mL
- Renewal rate of test solution: Semi-static exposure via the test medium. Reason: To increase constant exposure conditions both experiments of this study were conducted as static-renewal exposure. The renewal of the test solutions was done daily.
- No. of fronds per vessel: 10 (Exp. 1) and 11 (Exp. 2) fronds/test vessel at the start of exposure
- No. of vessels per concentration: 3 (Exp. 1) and 6 (Exp. 2)
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes; 20X AAP

TEST MEDIUM / WATER PARAMETERS
The test medium (20X AAP medium) was prepared as described in OECD Guideline for Testing of Chemicals, No. 221 Lemna sp. Growth inhibition Test, Mar 2006, Annex 4. Growth medium intended for testing was prepared 1–3 days before use to allow the pH to stabilize. The pH of growth medium was checked prior to use and readjusted if necessary, by the addition of 0.1 or 1 M NaOH or HCl. The test media was sterilized after preparation and prior to inoculation.

OTHER TEST CONDITIONS
- Test chamber: Infors HT Multitron Pro controlled climate cabinet.
- Test temperature: Temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels.
- Illumination: Artificial light; type universal white (OSRAM L 25), permanent illumination.
Light intensity:
Exp.1: 7003 - 7930 lux (mean: 7483 lux, coefficient of variation: 3.8%) at a wavelength of 400 - 700 nm.
Exp.2: 7563 - 8345 lux (mean: 7933 lux, coefficient of variation: 3.2%) at a wavelength of 400 - 700 nm.

TEST INITIATION AND MAINTAINANCE
Colonies consisting of 3 to 4 visible fronds were transferred from the inoculum culture and assigned to the test vessels under aseptic conditions. Each test vessel contained a total of 10 (Experiment 1) or 11 (Experiment 2) fronds. The number of fronds and colonies were the same in each test vessel. The test vessels were impartially distributed in an incubator at continuous illumination. Vessel positions were altered daily. The temperature was monitored within the incubator and in a separate vessel filled with deionized water. At the end of the test, the pH was measured in all replicates of the control and treatment groups; the pH at test initiation was measured in the respective bulk solution.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: on day 3, 5 and at exposure termination. Every frond visibly projecting beyond the edge of the parent frond was counted.
- Test endpoint parameter: Growth as frond number and dry weight
- Biomass: Dry weight was determined at the start of exposure in a comparable colony and at the end of exposure in all replicates. All colonies were collected from each of the test vessels and rinsed with deionized water. They were blotted to remove excess water and then dried at 60°C for 5 days to a constant weight. Any root fragments were included.
- Other: At the same time, observations on the appearance of the fronds included necrosis, chlorosis, changes in plant size or shape and root growth were documented.

RANGE-FINDING STUDY
Experiment 1 – Test with the complete dispersion:
The test concentrations were selected based on a range finding test (experimental conduct in accordance with GLP but without a GLP Status). The results of the 7 days range finding test were (as nominal concentrations):
ErC50 >100 mg/L
However, a 30% inhibition effect was observed on frond number at 100 mg/L. Therefore, a full concentration range was performed for Experiment 1.

Experiment 2 – Test with the dissolved fraction:
The test concentrations were selected based on the results of Experiment 1. A 22% inhibition effect was observed on frond number (based on mean growth rate data) at 100 mg/L (nominal). Experiment 2 was performed as limit study, in order to distinguish, whether the observed inhibition effect on fond number was triggered by the dissolved fraction or rather by the particulate fraction present in the complete dispersion applied in Experiment 1 of this study.
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorphenol (CAS 591-35-5)

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 0.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
dry weight
Remarks on result:
other: Experiment 1: complete dispersion
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
4.09 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
dry weight
Remarks on result:
other: Experiment 1: complete dispersion
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 69 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
dry weight
Remarks on result:
other: Experiment 1: complete dispersion
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
dry weight
Remarks on result:
other: Experiment 1: complete dispersion
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
4.57 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
dry weight
Remarks on result:
other: Experiment 1: complete dispersion
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
dry weight
Remarks on result:
other: Experiment 1: complete dispersion
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 1.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
dry weight
Remarks on result:
other: Experiment 2: dissolved fraction
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 1.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
dry weight
Remarks on result:
other: Experiment 2: dissolved fraction
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
dry weight
Remarks on result:
other: Experiment 2: dissolved fraction
Duration:
7 d
Dose descriptor:
NOELR
Effect conc.:
< 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
dry weight
Remarks on result:
other: Experiment 2: dissolved fraction
Duration:
7 d
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
dry weight
Remarks on result:
other: Experiment 2: dissolved fraction
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
dry weight
Remarks on result:
other: Experiment 2: dissolved fraction
Details on results:
EXPERIMENT 1 – Test with the complete dispersion:

see also Table 1, 2 and 3 for results

The duckweed population in the control vessels showed exponential growth, increasing from 10 fronds per vessel to an average of 317.2 fronds per vessel in the control after 7 days (corresponding to an average specific growth rate of 0.493 d-1). The dry weight increased from 1.8 mg (sample of the inoculum culture at start of exposure) to an average of 38.2 mg per vessel in the control at test termination.

The OECD 221 notes that toxicity values calculated by using the response variables average specific growth rate and yield are not comparable. The effect values based on average specific growth rate (ErCx)will be generally higher than those based on yield (EyCx). However, the differences are due to the mathematical basis of the calculations and should not be interpreted as a difference in sensitivity between the two variables. The results based on yield are
influenced by several factors including the Lemna species and clone, control growth rate as well as the slope of the concentration response curve. Results calculated based on average specific growth rate are independent of these variables and are the scientifically preferred basis to estimate toxicity. The preferred endpoint for this test is the lowest average specific growth rate ErC50 value.

The initially (0h) measured mean particle size distributions in the control and the lower test groups (0–3.2 mg/L) revealed a mean z-average in the range of 238–5998 nm, with a corresponding mean polydispersity index between 0.32–1.96, respectively. Along with an increasing z-average, the measured mean polydispersity index showed a positive correlation. The broad z-average range of the control and the mentioned test groups can most likely be considered as a result of the absence of particles (control) producing unreasonable and nonreliable results, as well as insufficient particle concentrations (nominal concentration 1 – 3.2 mg/L), leading to a weak scattering signal, aggravating the accuracy of the measurement. Likewise, the corresponding measurements after 2h and 24h old test solutions predominantly confirmed the trend of increasing particle sizes and polidisperity over time.
The test groups ≥10 mg/L revealed initial (0h) mean z-averages ranging from approx. 200–320 nm. The comparably narrow size range is accompanied by a mean polydispersity index between 0.14–0.37, respectively. Over the course of the 24h-long exposure intervals, the measured particle sizes tended to remain steadily over a period of up to 6h, shown by a more or less constant mean z-average and polydispersity index of the test substance dispersions, followed by distict increase of the latter two measured variables after 24h. These data point towards a reproducable initial dispersability of the test substance under testing conditions, which however, cannot be maintained over 24 h (for details see also non-GLP results of the OECD 318 Table 7).
Therefore, the test substance can be considered only shortly stable (here measured for a period of approx. 6h) when dispersed in the test medium under testing conditions. The increasing mean z-average over time seemingly resulted in sedimentation of the particulate test substance fractions over the 24-long reneval intervals towards the bottom of the test vessels, which can be confirmed by concentration control analyses, showing reduced test substance concentrations in the waterphase in the 24h old test dispersions, relative to the 0h freshly prepared test dispersions.


EXPERIMENT 2 – Test with the dissolved fraction:

see also Table 4, 5 and 6 for results

The duckweed population in the control vessels showed exponential growth, increasing from 11 fronds per vessel to an average of 349.3 fronds per vessel in the control after 7 days (corresponding to an average specific growth rate of 0.494 d-1). The dry weight increased from 1.2 mg (sample of the inoculum culture at start of exposure) to an average of 42.4 mg per vessel in the control at test termination.

No particle size was measured, as the particulate fraction was removed from the test solutions. Only the dissolved fraction was tested in Experiment 2.

Results with reference substance (positive control):
The EC50 (7d, based on frond number) of the reference substance 3,5-Dichlorphenol was 7.86 mg/L (for Experiment 1, experiment date: 12 Jun 2020, project number: 63E0308/02E014), and 8.46 mg/L (for Experiment 2, experiment date: 20 Nov 2020, project number: 63E0308/02E014) These results are within the range of 5.9 – 13.3 mg/L and indicates that the culture of Lemna gibba used in this study is responding normally to toxic stress.

Any other information on results incl. tables

Experiment 1 – Test with the complete dispersion:


 


Table 1: Mean specific growth rate inhibition of Lemna gibba in each test group relative to the Control:




























































Nominal concentration [mg/L]

Average specific growth rate [d-1] inhibition over 7-days



Frond number, mean (%CV)



 


% Inhibition



Dry weight, mean (%CV)



 


% Inhibition



0 (control)



0.493 (2.6)



0%



0.434 (2.7)



0%



1



0.472 (1.4)



4.4



0.413 (2.1)



4.8



3.2



0.460 (3.2)



6.8



0.396 (3.3)



8.6



10



0.448 (3.6)



9.2



0.374 (4.2)



13.7



32



0.419 (3.1)



15.0



0.334 (2.8)



22.9



100



0.386 (1.0)



21.9



0.289 (1.5)



33.3



Inhibition greater than 100% or lower than 0% were replaced by 100% and 0%, respectively.



 


Table 2: Lemna Morphology and visual signs of Phytotoxicity



















































































































































Nominal concentration [mg/L]ReplicateMorphological changes, signs of phytotoxicity
Day 3Day 5Day 7
01000
2000
3000
4000
5000
6000
11006
2006
3006
3.21008+
2008+
3008+
1018+8++, 1+8++, 1+, 3+
28+8++, 1+8++, 1+, 3+
38+8++, 1+8++, 1+, 3+
3218++1+, 4+, 7+, 8++1+, 4+, 7+, 6+, 8++, 3+
28++1+, 4+, 6+, 8++1+, 4+, 7+, 6+, 8++, 3+
38++1+, 4+, 6+, 8++1+, 4+, 7+, 6+, 8++, 3+
10018++, 1+1++, 4++, 6+, 8+++1++, 4++, 6++, 7+, 8+++, 3++
28++, 1+1++, 4++, 6++, 8+++1++, 4++, 6++, 7+, 8+++, 3++
31+, 6+, 8+++1++, 4++, 6++, 7+, 8+++1++, 4++, 6++, 7+, 8+++, 3++

Explanation of abbreviations:                   


0:         no findings


1:         frond or root yellowing (chlorosis)


2:         fronds humped or swollen (gibbosity)


3:         small fronds


4:         shortened roots


5:         no roots


6:         plants fragmented (fronds and/or roots)


7:         dead white plant tissue (necrosis)


8:         plants colored by test substanz (fronds and/or roots)


(+):       slight effects


(++):    medium effects


(+++):  strong effects


 


Table 3: Dissolution Behaviour






































































 


Nominal concentration [mg/L]



day 0



day 1



day 2-7



fresh



fresh



old



fresh



old



0 (control)



0



0



0



0



0



1



1



1



1



1



1



3.2



1



1



1



1



1



10



2



2



2



2



2



32



2



2



2



2



2



100



2



2



2



2



2



Explanation of abbreviations:


0:         clear, colorless test medium


1:         coloration caused by the test substance


2:         red dispersion, coloration caused by the test substance



 


Experiment 2 – Test with the dissolved fraction:


 


Table 4: Mean specific growth rate inhibition of Lemna gibba in each test group relative to the Control

































 


Loading rate [mg/L]



Average specific growth rate [d-1] inhibition over 7-days



Frond number, mean (SD)



 


% Inhibition



Dry weight, mean (SD)



 


% Inhibition



0 (control)



0.494 (0.010)



0%



0.509 (0.008)



0%



100



0.461 (0.009)



6.6%



0.478 (0.005)



6.0%



Inhibition greater than 100% or lower than 0% were replaced by 100% and 0%, respectively.



 


Table 5: Lemna Morphology and visual signs of Phytotoxicity

























































































Nominal concentration [mg/L]ReplicateMorphological changes, signs of phytotoxicity
Day 3Day 5Day 7
01000
2000
3000
4000
5000
6000
1001000
2000
3000
4000
5000
6000

Explanation of abbreviations:                        


0:         no findings


1:         frond or root yellowing (chlorosis)


2:         fronds humped or swollen (gibbosity)


3:         small fronds


4:         shortened roots


5:         no roots


6:         plants fragmented (fronds and/or roots)


7:         dead white plant tissue (necrosis)


8:         plants colored by test substanz (fronds and/or roots)


(+):       slight effects


(++):    medium effects


(+++):  strong effects


 


Table 6: Dissolution Behaviour



























 


Loading rate [mg/L]



day 0-7



fresh



old



0 (control)



0



1



100



1



1



Explanation of abbreviations:


0:         clear, colorless test medium


1:         coloration caused by the test substance



 


Table 7: Full results of the organic pigment dispersion stability in the OECD 201, 211 and 221 media.











































Pigment NameTest MediumStability after 6h, %Standard deviation, %Stability after 15h, %Standard deviation, %Stability after 24h, %Standard deviation, %
Pigment Red 53:1OECD 21098.40.195.80.293.20.7
OECD 21175.70.944.72.524.32.3
OECD 221950.476.74.259.53.5

 


 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test substance is acutely not harmful for aquatic plants. And, based on long-term (chronic) toxicity study data, the product is very likely not harmful to aquatic plants.