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Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
The following test vessels were set up:
• Test solution (Tn); containing test water, test item and duckweed (three replicates per concentration)
• Blank control (Bn); containing test water and duckweed (six replicates)
Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution (test item dissolved in test water) with test water. The resulting solutions were used as the test solutions in the toxicity test.
Pure test water served as blank controls
Vehicle:
yes
Remarks:
pure water
Details on test solutions:
The following test vessels were set up:
• Test solution (Tn); containing test water, test item and duckweed (three replicates per concentration)
• Blank control (Bn); containing test water and duckweed (six replicates)

Since the test item is soluble, the test solutions were prepared by respective dilutions of a stock solution with test water. The resulting solutions were used as the test solutions in the toxicity test.
Pure test water served as blank controls.
Young and rapidly growing plants without visible lesions or discoloration (chlorosis) from an exponentially growing monoculture of Lemna minor were distributed into the test vessels (9-12 fronds per replicate, 2-4 fronds per plant).
The vessels were randomly placed in the incubation chamber at the start of the test and randomly repositioned after the observations made at days 3 and 5.

Samples were taken from all test solutions including the control at the beginning of the test and after 3, 5 and 7 days of exposure. For sampling at day 3, 5 and 7, the replicates per treatment were pooled.
Samples were not filtered or centrifuged.
All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions.

Photometric determinations of the test concentrations were performed with a Shimadzu UV-1800 photometer (Shimadzu Schweiz GmbH, Römerstr. 3, CH-4153 Reinach) at a wavelength of 390 nm.

A validation of the photometric method was conducted in compliance with GLP during this study (calibration series followed by 10 measurements of a control standard at a concentration of 12.2 mg/l). The following validation parameters were derived:
• Goodness of fit: 1.000 (R2 of the linear calibration regression)
• Repeatability: 0.45% (Relative Standard Deviation of the ten measurements)
• Accuracy: 98% (mean of the ten measurements relative to the expected value)
• Limit of quantification (LOQ) as determined in the present study: 2.23 mg/l test item, corresponding to 1.12 mg/l a.i.; the LOQ is based on the lowest standard solution measured of 2.23 mg/l.
Test organisms (species):
Lemna minor
Details on test organisms:
The test organism used for the study was Lemna minor supplied by the German Environment Office (Umweltbundesamt, FGIII 2.5, Überwachungsverfahren Abwasserentsorgung, Schichauweg 58, D- 12307 Berlin.

For evaluation of the duckweed quality and the test procedure, 3,5-dichlorophenol is tested as a positive control at least once per year. The result of the positive control test performed in June 2019 showed that the sensitivity of the test system based on growth rate of the frond number was in line with the recommended 1.8–3.6 mg/l (according to guideline ISO/CD 20079 “Determination of the toxic effect of water constituents and waste water to duckweed (Lemna minor) — Duckweed growth inhibition test”).

Pre-culture:
Exponentially growing plant monoculture of Lemna minor; healthy plants with 2-4 fronds were used; the culture was visibly free from contamination by other organisms such as algae and protozoa
Test type:
static
Water media type:
other: ultra pure water
Limit test:
no
Total exposure duration:
7 d
Test temperature:
24±2 °C
pH:
6.5±0.2 at the beginning of the test; the pH value in the blank ontrol should not increase by more than 1.5 units during the test
Nominal and measured concentrations:
Prior to the definitive test a non-GLP range finding test with nominal concentrations of 10, 100 and 500 mg/l of Direct Yellow 50’s active ingredient was performed (3 replicates for the blank controls and 2 replicates per test concentration).

Based on the results of this non-GLP range finding test, the following nominal concentrations were chosen for the definitive test1: 992, 314, 99.2, 31.4 and 9.92 mg/l of the test item, corresponding to 500, 158, 50.0, 15.8, and 5.00 mg/l of the active ingredient.
Details on test conditions:
Test vessel:
400 ml beakers, all-glass, with 200 ml of test water. The beakers were covered with black paper up until the 200 ml mark to ensure that illumination comes only from above and not from the sides.

Pre-culture:
Exponentially growing plant monoculture of Lemna minor; healthy plants with 2-4 fronds were used; the culture was visibly free from contamination by other organisms such as algae and protozoa.

Incubation:
The beakers were incubated on a black non-reflecting surface in a climatic chamber from Memmert, type IPP260 (Memmert GmbH+Co.KG, D-91123 Schwabach FRG).

Illumination Supplied by fluorescent tubes from Romberg, type PAR white, T5HO 55 cm, 24-Watt, 6’400 Kelvin (Romberg & Sohn GmbH+Co KG, D-25479 Ellerau).
Photoperiod: 24h.
Intensity: 6500–10000 lux
Homogeneity: within ±15%
Reference substance (positive control):
yes
Remarks:
pure test water
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
Test concentrations
The test concentrations of the test item during the 7-day test period were determined by photometry at the beginning of the test as well as after 3, 5 and 7 days of exposure. These analyses confirmed the right dosage of the test item and showed that the concentrations were satisfactorily maintained over the 7-day test period. The measured concentrations of the active ingredient were 468, 147, 45.6, 14.3 and 4.37 mg/l at the beginning of the test and 460, 139, 42.4, 13.7 and 4.12 mg/l (i.e. 98, 95, 93, 96% and 94% of the initial value, respectively) at the end of the test. All concentrations remained within 80-120% of the nominal value.
Therefore, the effective concentrations ECx were assessed based on the nominal concentration of the active ingredient, which were 500, 158, 50.0, 15.8 and 5.00 mg/l.

Effects on frond number
With respect to both endpoints growth rate and yield inhibition, no significant dose-response relationship was found. Also, no significant effects (Dunnett’s test, one-sided smaller,  = 0.05) were observed compared to the untreated controls.
Based on these observations and the nominal concentrations of the active ingredient, the median effect concentration (EC50) with respect to the frond number’s growth rate and yield to Lemna minor was estimated to be >500 mg/l and the NOEC was determined to be 500 mg/l.

Effects on dry weight
With respect to growth rate the following effects as compared to the untreated controls were observed: 25% at 500 mg/l. No significant effects were observed at 158, 50.0, 15.8 and 5.00 mg/l nominal a.i. concentrations. No significant dose-response relationship was found.
Based on these data and the nominal concentration of the a.i., the 7-day EC50 with respect to the dry weight’s growth rate was estimated to be >500 mg/l.
The LOEC with respect to the average dry weight’s growth rate was determined to be 500 mg/l according to a Williams’ multiple t-test (one-sided smaller,  = 0.05). The corresponding NOEC is therefore 158 mg/l.
With respect to yield the following effects as compared to the untreated controls were observed: 42% at 500 mg/l. No significant effects were observed at 158, 50.0, 15.8 and 5.00 mg/l nominal a.i. concentrations. No significant dose-response relationship was found.
Based on these data and the nominal concentration of the a.i., the 7-day EC50 with respect to the dry weight’s yield was estimated to be >500 mg/l.
The LOEC with respect to the average dry weight’s yield was determined to be 500 mg/l according to a Williams’ multiple t-test (one-sided smaller,  = 0.05). The corresponding NOEC is therefore 158 mg/l.

Environmental conditions
The pH value in the control drifted by 1.0 unit during the whole test period. The light intensity was 8470-9630 lux (mean 8955 lux; max. variation ±7.5%) at the start of the test and 8110-9800 lux (mean 8881 lux, max. variation ACH ±10.3%) at the end of the exposure period. All values were within the guideline ranges.
The test media did not show any signs of precipitation, at any of the test concentrations

The appearance of the plants (number and size of the fronds and roots) at the end of the test is summarized in the following table:

Nominal concentration of active ingredient [mg/l]

 Fronds [Colour, size]  Roots [lenght]
 Control Lush green; ca 3 -5 mm average size

Reaching to the bottom of the test vessel with some shorter ones

 5.00 Lush green; ca 3 -5 mm average size  

Reaching to the bottom of the test vessel with some shorter ones

 
 15.8 Lush green; ca 3 -5 mm average size   

Reaching to the bottom of the test vessel with some shorter ones

 50.0 Lush green; ca 3 -5 mm average size   

Reaching to the bottom of the test vessel with some shorter ones

 158 Lush green; slightly smaller than control   

Reaching to the bottom of the test vessel with some shorter ones

 500 Lush green; smaller than control, mostly 1/2 the size of control  Partly shorter roots than control

The smaller leaves in the higher concentrations explain why significant effects could be observed based on dry weight but not based on frond number

Validity criteria fulfilled:
yes
Remarks:
Validity criterion: Doubling time of the frond number in the control 1.95 (required value: ≤2.5)
Conclusions:
The 7-day EC50 (based on the frond number's growth rate) of test item on the on the duckweed Lemna minor is >500 mg/l.
Executive summary:

The growth inhibitory effects of the test item to the duckweed Lemna minor were investigated according to OECD guideline 221, under static conditions over a period of seven days.

The test solutions were prepared by appropriate dilutions of a stock solution in test water.

The nominal concentrations were 992, 314, 99.2, 31.4 and 9.92 mg/l of the test item, corresponding to 500, 158, 50.0, 15.8, and 5.00 mg/l of the active ingredient. Three parallel test vessels were used for each concentration of the test item and six vessels for the controls.

The test concentrations of the test item during the 7-day test period were determined by photometry at the beginning of the test, as well as after 3, 5 and 7 days of exposure. These analyses showed that the test concentrations were correctly dosed and that they remained stable over the seven-day test period. Since all concentrations were within 80–120% of the nominal value the effective concentrations ECx were assessed based on the nominal concentrations of the active ingredient.

The 7-day EC50 (based on the frond number’s growth rate) of the test item on the duckweed Lemna minor is >500 mg/l.

This value is based on the nominal concentrations of the active ingredient.

The validity criterion was fulfilled

Description of key information

The 7-day EC50 (based on the frond number's growth rate) of test item on the on the duckweed Lemna minor is >500 mg/l.

Key value for chemical safety assessment

Additional information