mancozeb (ISO); manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt  

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:

2009-12-04 to 2010-04-16

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:

adopted March 2006

Deviations:

yes

Remarks:

see remarks in section "Principles of method if other than guideline"

Principles of method if other than guideline:

On two occasions during the definitive test, the maximum temperature of the incubator slightly exceeded that recommended for this type of study (26ºC).
On the final two days of the test the temperature of the incubator was not recorded in error.

On the last recorded occasion and the day after the test ended, the temperature of the incubator was slightly over range so it was uncertain if the temperature was over range when not recorded. The temperature of the media measured during the test was within acceptable limits.

As the validity criterion for the study were achieved these deviations were not considered significant or to have affected the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

During the definitive test, two samples (5 mL) of the freshly-prepared control and test media were taken from preparation flasks on Days 0 and 5; samples of expired media were collected from the pooled contents of the replicate control and test vessels on Days 3 and 7. Additional samples of expired media were taken from flasks containing Mancozeb 80 WP at 4.44 mg/L but with no Lemna on Days 3 and 7, in order to obtain information on the stability of the test substance in the presence and absence of the plants. On each occasion, one sample at each concentration was analysed and the other was stored in a freezer in case further analysis was required. Samples from the test were hence analysed in suitably sized batches along with control samples fortified with Mancozeb 80 WP which acted as procedural recovery samples.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: On each occasion of media preparation, the test substance was dispersed in culture medium (1 L) in a volumetric flask to give a stock concentration of 100 mg/L as product. The contents of the flask was shaken and treated with ultrasound. Aliquots of the stock (455 to 1.3 mL) were then diluted with SIS medium to give the required test concentrations.
- Controls: blank control
- Evidence of undissolved material: no

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: common duckweed
- Strain: not specified
- Source: laboratory culture originating from specimens obtained from University of Toronto Culture Collection (UTCC), Canada.

ACCLIMATION
Cultures of Lemna were initiated at least seven days prior to the start of the definitive test. These cultures were grown in fresh sterile nutrient medium (Appendix 2) and acclimatised to test conditions in a controlled environment area under continuous illumination. During the culturing period and prior to the initiation of the test, the light levels were maintained between (nominally) 6500 to 10000 lux and the temperature was maintained nominally at 24 ± 2 °C.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24 ± 2 °C

pH:

ranging between 6.86 (freshly prepared media) to 10.38 (expired media)

Dissolved oxygen:

not specified

Nominal and measured concentrations:

Nominal: 0 (control), 105, 346, 1110, 3570, 11400 and 36600 µg a.s./L;
Measured: freshly prepared media 0 (control), 8.14, 24.6, 67.3, 426, 3403 and 4075 µg a.s./L; expired media contained about 11% of nominal values

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: no (temperature controlled incubator)
- Test vessel: conical flasks
- Type: open
- Material fill volume: material not specified, 500 mL nominal volume
- Aeration: no
- Agitation: no
- Renewal rate of test solution: On Days 3 and 5 of the test, all plants were carefully transferred to clean sterile vessels containing freshly-prepared control or test media in an attempt to maintain stable exposure levels.
- No. of fronds per vessel: twelve
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: 6 x 30 W “cool white” 1 metre fluorescent tubes with a nominal lux of 6500 to 10000

EFFECT PARAMETERS MEASURED: At each media renewal and at the end of the test, all plants were examined and any differences in growth habit or general health in comparison to the control were noted. The occurrence of frond deformation, chlorosis or necrosis and changes in root length were also recorded along with any other abnormalities and the number of plants. Frond numbers were counted in each control and test vessel on Days 3, 5 and 7 of the exposure period as an assessment of growth. Growth was monitored after 3, 5 and 7 days by counting the total number of fronds in each vessel. At termination of the study the dry weight of the plants was determined.
- Determination of frond number: manual counting
- Determination of biomass: dry weight

RANGE-FINDING STUDY
The range finding test employed nominal test concentrations of 0.01, 0.1, 1.0 and 10 mg/L (as product). No inhibition occurred at 0.01, 0.1 and 1 mg/L. At 10 mg/L, the average specific growth rate of the fronds was inhibited by 54%. Based on these results, a nominal concentration range of 0.880, 1.94, 4.27, 9.39, 20.7 and 45.5 mg/L (as product) were selected but this definitive test did not provide a NOEC for any of the test parameters therefore a second definitive test was conducted using the following nominal test concentrations; 0.130, 0.430, 1.38, 4.44, 14.2 and 45.5 mg/L as product (0.105, 0.346, 1.11, 3.57, 11.4 and 36.6 mg/L as Mancozeb).

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

117 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

1 042 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

growth rate

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

37.1 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

24.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

frond number

growth rate

yield

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

24.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

growth rate

yield

Details on results:

No abnormalities were observed in the controls and at test concentrations of 0.130 and 1.38 Mancozeb 80 WP/L. At 0.43 mg Mancozeb 80 WP/L, pale and chrorotic fronds were observed. As well as these abnormalities, short roots were seen from 4.44 mg Mancozeb 80 WP/L, small fronds were noted from 14.2 mg Mancozeb 80 WP/L, and colony break up was observed at 45.5 mg Mancozeb 80 WP/L. The control and test media (fresh and expired) from 0.130 to 4.44 mg Mancozeb 80 WP/L were colourless except on Day 7 when they were green hazy dispersions. The media at 14.2 and 45.5 mg Mancozeb 80 WP/L were hazy/opaque yellow coloured dispersions.

VALIDITY OF STUDY
The doubling time of frond number in the control cultures was to have been less than 2.5 days (60 hours), corresponding to approximately a seven-fold increase in seven days. This criterion was fulfilled and the study is hence regarded valid.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

The data was compiled in an Excel spreadsheet and analysed using SAS 9.1 (SAS Institute 2002). Test results were expressed in terms of the nominal product concentrations and mean measured concentrations of mancozeb in samples of fresh media taken on Days 0 and 5. In order to estimate the concentration at which 5 %, 50 % and 90 % inhibition of growth occurred (EC5, EC50 and EC90), sigmoidal curves were fitted to AUCP and growth rate. Values for biomass were determined accordingly. All 95 % confidence intervals for ECP were calculatedusing the likelihood ratio method (Donaldson and Schnabel, 1985). Each of the treated groups was compared with the control groups using a two-sided Williams test for monotonic trend to determine the LOEC and the NOEC for each parameter.

Table 1: Analytical data and results of their re-evaluation (RAR Mancozeb, 2018)

Time [d]	Nominal concentration [µg a.s./L]
	103	346	1110	3570	11400	36600
	Measured concentration [µg a.s./L]
0	51.9	334	599	2480	8300	28300
3	0.8*	5**	6.72	80.3	1060	1990
5	132	275	1060	3640	12000	36000
7	0.8*	0.8*	4.82	45.4	1270	136
Geomean	8.14	24.6	67.3	426	3403	4075
LOD: 0.8 µg a.s./L, LOQ: 10 µg a.s./L * not detected; LOD was used instead ** < LOQ; LOQ/2 was used instead

 

Table 2: ECx values based on geometric mean measured concentrations (re-evaluation, RAR Mancozeb, 2018)

Endpoint	Effect concentration [µg a.s./L] (95% confidence intervals)
	NOEC	LOEC	EC10	EC20	EC50
Frond Number
Growth rate	24.6	67.3	82.2 (38.6 - 175)	238 (115 - 494)	1811 (730 - 4370)
Yield	24.6	67.3	38.4 (16.4 – 90.0)	86.1 (38.3 - 192)	403 (156 - 1057)
Sectional growth rate	24.6	67.3	26.3 (5.75 – 120)	76.1 (17.9 – 319)	583 (106 – 3270)
Biomass
Growth rate*	24.6	67.3	37.1 (13.1 – 105)	117 (43.3 – 313)	1042 (312 – 3425)
Yield	24.6	67.3	16.0 (5.10 – 50.5)	36.9 (12.4 – 109)	184 (49.5 – 676)
* covering sectional growth rate

 

Validity criteria fulfilled:

yes

Conclusions:

For growth rate the 7-day ErC50 and ErC10 were 1811 μg a.s./L and 82.2 μg a.s./L (mean measured) based on frond numbers. The 7-day ErC50 and ErC10 based on biomass were determined to be 1042 μg a.s./L and 37.1 μg a.s./L (mean measured).
Based on yield, the 7-day EyC50 and EyC10 were 403 μg a.s./L and 38.4 μg a.s./L (mean measured) based on frond numbers, and 184 μg a.s./L and 16.0 μg a.s./L (mean measured) for biomass.
The No-Observed-Effect Concentration (NOEC) established during this study was 24.6 µg a.s./L (mean measured) for all investigated endpoints (growth rate and yield for frond number and biomass).

Executive summary:

A 7-day study on the toxicity of Mancozeb 80 WP (purity: 80.5% Mancozeb) to the aquatic plant Lemna minor was performed to OECD guideline number 221 and in compliance with GLP principals. Six replicate vessels were prepared for each control group and three vessels for each treatment group. Each initially contained twelve fronds of Lemna. The plants were maintained in a temperature controlled incubator under continuous illumination.

Exposure to the test item was in semi-static conditions at nominal formulation concentrations of 0 (control), 0.13, 0.43, 1.38, 4.44, 14.2, and 45.5 mg Mancozeb 80 WP/L. Mean measured concentrations in fresh media samples taken on days 0 and 5 were 0 (control), 0.0828, 0.303, 0.797, 3.00, 9.98, and 31.9 mg a.s./L, which related to 72-88% of nominal concentrations. Mean measured levels in the spent media samples taken on days 3 and 7 were 0 (control), 0, 0, 0.0057, 0.06, 1.16, and 0.52 mg a.s./L, relating to 0-8% of nominal values. Based on the geometric mean following concentrations were used for endpoint calculations: 0 (control), 8.14, 24.6, 67.3, 426, 3403 and 4075 µg a.s./L.

 

No abnormalities were observed in the controls and at test concentrations 8.14 and 24.6 µg a.s./L (mean measured). For growth rate the 7-day ErC50 and ErC10 were 1811 μg a.s./L and 82.2 μg a.s./L (mean measured) based on frond numbers. The 7-day ErC50 and ErC10 based on biomass were determined to be 1042 μg a.s./L and 37.1 μg a.s./L (mean measured).

Based on yield, the 7-day EyC50 and EyC10 were 403 μg a.s./L and 38.4 μg a.s./L (mean measured) based on frond numbers, and 184 μg a.s./L and 16.0 μg a.s./L (mean measured) for biomass.

The No-Observed-Effect Concentration (NOEC) established during this study was 24.6 µg a.s./L (mean measured) for all investigated endpoints (growth rate and yield for frond number and biomass). All validity criteria were fulfilled.

 

The key-value of 1042 µg a.s./L based on the ErC50 of biomass is considered for the risk assessment.

Description of key information

In a semi-static Higher Plant Toxicity Test according to OECD TG 221 with L. minor a 7-day ErC50 of 1042 μg a.s./L (mean measured) was determined. The overall NOEC of 24.6 µg a.s./L (mean measured) was established.

Key value for chemical safety assessment

EC50 for freshwater plants:

1 042 µg/L

EC10 or NOEC for freshwater plants:

24.6 µg/L

Additional information

The summary below was taken from the CHL report of Mancozeb (De 2017): A 7-day study on the toxicity of Mancozeb 80 WP (purity: 80.5% Mancozeb) to the aquatic plant Lemna minor was performed to OECD guideline number 221 and in compliance with GLP principals. Exposure to the test item was in semi-static conditions at nominal formulation concentrations of 0 (control), 0.13, 0.43, 1.38, 4.44, 14.2, and 45.5 mg Mancozeb 80 WP/L. Mean measured active concentrations in fresh media samples taken on days 0 and 5 were 0 (control), 0.0828, 0.303, 0.797, 3.00, 9.98, and 31.9 mg a.s./L. These related to 72 - 88% of nominal concentrations. Mean measured levels in the spent media samples taken on days 3 and 7 were 0 (control), 0, 0, 0.0057, 0.06, 1.16, and 0.52 mg a.s./L, relating to 0 - 8% of nominal values. The mean measured (fresh media) ErC50 was 9.56 mg a.s./L. The RMS requested endpoints based on mean measured values of fresh and expired media samples. The applicant provided these values which were calculated to be: ErC50 (fronds number) = 1811 μg a.s./L (mm), ErC10 (fronds number) = 82.2 μg a.s./L (mm), ErC50 (biomass) = 1042 μg a.s./L (mm), and ErC10 (biomass) = 37.1 μg a.s./L (mm). These values were considered acceptable for use in the risk assessment for the AIR 3 renewal of Mancozeb as a pesticide and also for hazard classification.