Registration Dossier

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:
29/02/08- 09/07/08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study conducted to current guideline. The read-across from MnCl2 to MnSO4 is justified on the following basis: both substances are very soluble in water hence bioavailable and both will release Mn2+ ions. Therefore, from an ecotoxicity standpoint, the chloride or sulphate anions are not considered to have any influence on the effective toxicity of Mn2+ or any toxicity in their own right, so the anions can be disregarded. Therefore any effect will be related to the Mn2+ cation, and the data from MnCl2 ecotoxicity tests is regarded as a suitable surrogate for read-across.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
U.S. EPA GLP standard (40 CFR 792)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Total recoverable and dissolved (filtered through a 0.45 µm nylon mesh) manganese were detmined at test initiation and termination. Also dissolved Manganese was monitored on days 3 and 5.
- Sampling method: Media samples from each treatment were collected for determination of manganese at test initiation. Samples for dissolved manganese concentration were collected from the new renewal waters within each treatment on days 3 and 5. At test termination, samples were taken from replicate A for each concentration. Metal samples (~ 20 mLs) were taken from the centre of the test using a 30-mL syringe.
- Sample storage conditions before analysis:Samples were preserved with nitric acid (trace metal grade) to pH <2 prior to analysis

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was added to laboratory blended water to make a stock solution of 50 g Mn/L. The concentrations were prepared by adding the appropriate volume of stock solution to the growth media.
- Controls: dilution water.

Test organisms

Test organisms (species):
Lemna minor
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Obtained from the University of Toronto culture and collection (UTCC; Toronto, ON, Canada). This strain was collected by Barabara E. Giles in 1977 from Wainfleet, Stinking Barn, Niagara Peninsula, Ontario, Canada.
- Age of inoculum (at test initiation): 9 Days old

Study design

Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
7 d
Post exposure observation period:
Not reported

Test conditions

Hardness:
52 mg/L as CaCO3
Test temperature:
24±2°C
pH:
6.8-7.6
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
3.88, 7.75, 15.5, 31 and 62 mg Mn/ L - Nominal concentration
<0.05, 3.35, 7.01, 15.57, 30.72 and 64.94 mg Mn/ L - Average measured dissolved concentration
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL beakers
- Type (delete if not applicable): open
- Fill volume: Holds approximately 200 mL of test solution
- No. of colonies per vessel: 9- 12 fronds
- No. of fronds per colony: 2- 4 visible fronds
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):3

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Test was conducted in a synthetic freshwater reconstituted from deionised water with the addition of appropriate reagent grade salts.
- Total organic carbon: 0.7 mg/ L (DOC = dissolved organic carbon. Measured via combustion (EPA 415.1; CH2M HILL, Corvallis, OR)
- Chlorine:20.5 mg/L (Measured via Ion Chromatography (EPA 300.0; CH2M HILL, Corvallis, OR)
- Alkalinity: 12 mg/L as CaCO3
- Conductivity:270 µS/ cm
- Intervals of water quality measurement: Hardness, alkalinity, total residual chlorine, and ammonia were measured in the dilution water at test initiation only. Temperature within the environmental chamber were recorded daily. Test solution pH was measured at test initiation on renewal days ( days 3 and 5) in both new and old waters.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod:Continous
- Light intensity and quality: 600 -930 foot candles.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting, computerized image analysis and examined for grossly observable abnormalities.
- Determination of biomass: dry weight. The colonies from each replicate were placed into a pre- weighed pan and dried at 60°C for 72 hours and then re-weighed to the nearest 0.01 mg to obtain a dry weight.
- Other: Normal and abnormal appearing fronds were determined at the beginning of the test, and on at least two occasions during the test. Changes in plant development (e.g. frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy), and in root length and appearance was recorded.

RANGE-FINDING STUDY
- Test concentrations: 31 mg Mn/L and higher
- Results used to determine the conditions for the definitive study: The test concentrations were selected based on findings from a range-finder test that resulted in effects on growth at treatments at 31 mg Mn/L and higher.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
30.72 other: mg Mn/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
other: Number of fronds, total dry weight and growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
64.94 other: mg Mn/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
other: Number of fronds, total dry weight, and growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
23.37 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
frond number
Remarks on result:
other: 95% CL
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
26.44 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
other: Total dry weight
Remarks on result:
other: 95% CL
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
41.54 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: 95% CL
Details on results:
Not reported
Results with reference substance (positive control):
Not reported
Reported statistics and error estimates:
All statistical analyses were performed using the measured average dissolved manganese concentrations. Difference in growth (based on frond count, dry weight, biomass and growth rate) were evaluted using statistical computer packages (TRAP, U.S. EPA Duluth, MN, USA, and CETIS, Tidepool Scientific Software Mckinleyville, CA, USA). Since the data met the assumptions of normality and homogeneity, the NOEC and LOEC were estimated using an analysis of variance (Dunnett's) to compare (p=0.05) organism performance in the experimental treatments with the observed in the control. The EC10 was estimated using a piecewise linear regression. Exposure concentrations were log-transformed before determination of EC10 values.

Any other information on results incl. tables

 Table 5. Frond Count Data

 

Average Measured Dissolved Concentration.(mg Mn /L)

Total Number of Fronds (% abnormal)

Sig. Less than the Control1

Rep A

Rep B

Rep C

Average(±SD)

< 0.05

257

290

253

267±20

-

3.34

226

288

304

273±41

No

7.01

182(1)

250(0.4)

248(0.4)

227±39

No

15.57

273(3)

252

254(3)

260±12

No

30.72

233(8)

217

290(14)

247±38

No

64.94

172(49)

146

181(49)

166±18

Yes

1Significantly less than the control (p =0.05) using Dunnett’s Test.

SD = Standard Deviation

 

Table 6. Total Dry weight

 

Average Measured Dissolved Concentration.(mg Mn /L)

Dry weights (units)

Sig. Less than the Control1

Rep A

Rep B

Rep C

Average(±SD)

< 0.05

24.82

29.56

23.80

26.06±3.07

-

3.34

24.57

31.82

33.18

29.86±4.63

No

7.01

20.25

25.91

24.40

23.52±2.93

No

15.57

30.75

26.12

25.37

27.41±2.91

No

30.72

23.60

19.33

22.83

21.92±2.28

No

64.94

11.12

10.46

12.46

11.35±1.02

Yes

1Significantly less than the control (p =0.05) using Dunnett’s Test.

SD = Standard Deviation

 

Table 7.Growth Rate from Day 0 to Day 7

 

Average Measured Dissolved Concentration.(mg Mn /L)

Dry weights (units)

Sig. Less than the Control1

Rep A

Rep B

Rep C

Average(±SD)

< 0.05

5.24

5.36

5.22

5.27±0.08

-

3.34

5.11

5.35

5.40

5.29±0.16

No

7.01

4.88

5.20

5.20

5.09±0.19

No

15.57

5.27

5.20

5.20

5.22±0.04

No

30.72

5.05

4.99

5.20

5.08±0.11

No

64.94

4.15

4.00

4.22

4.13±0.11

Yes

  

Table 8.Summary of Test Statistics (mg Mn/ L)1

Evaluation

Frond Count

Total Dry Weight

Growth Rate

NOEC

30.72

30.72

30.72

LOEC

64.94

64.94

64.94

EC10 (95 % CI)

23.37

(11.67-46.78)

26.44

(18.04- 38.74)

41.54

(37.37- 46.17)

EC20 (95% CI)

35.07

(21.22-57.94)

31.94

(23.59-43.26)

62.48

(54.96-71.04)

 1NOEC = No observed effect concentration. LOEC = Lowest observed effect concentration. EC10= 10% Effect concentration. EC50 = Median effect concentration. CI= Confidence intervals.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The doubling time of frond number in the control was less than 2.5 days (60h).
Conclusions:
The 7 day NOEC was 30.72 mg Mn/L. The read-across from MnCl2 to MnSO4 is justified on the following basis: both substances are very soluble in water hence bioavailable and both will release Mn2+ ions. Therefore, from an ecotoxicity standpoint, the chloride or sulphate anions are not considered to have any influence on the effective toxicity of Mn2+ or any toxicity in their own right, so the anions can be disregarded. Therefore any effect will be related to the Mn2+ cation, and the data from MnCl2 ecotoxicity tests is regarded as a suitable surrogate for read-across.