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Ecotoxicological information

Long-term toxicity to fish

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Endpoint:
fish early-life stage toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
See the read-across report attached in Section 13.
Cross-reference
Reason / purpose:
read-across source
Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
03/06/08 - 08/07/08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Remarks:
Study conducted to GLP and to a 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.
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose:
other: Read-across target
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
U.S. EPA Good Laboratory Practise (GLP) standard (40 CFR 792)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Analytical samples from each treatment were collected for total recoverable and dissolved (filtered through a 0.45 µm nylon mesh) Mn analysis at test initiation. For the remainder of the test, samples for dissolved Mn analysis were collected from alternating replicates within each treatment twice a week. Metal samples (~ 15 mL) were taken from the centre of the test chamber (at a depth of approximately 2 inches) using a 20-mL syringe. All samples was analysed via Flame atomic absorption spectrophotometry (AAS).
- Sample storage conditions before analysis:Samples were preserved with nitric acid (trace metal grade) to pH <2 prior to analysis
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Manganese (II) chloride was added to deionized water to achieve a nominal stock solution concentration of 192 mg Mn/L. A Marriotte bottle was used to continuously deliver an appropriate volume of Mn stock solution to the diluter. Stock solution was delivered to the dilutor at a rate of 5.0 mL/ minute, checked daily, and adjusted as needed. A new stock solution was prepared every other day during the test. The calibration of the diluter was checked prior to the test, and weekly thereafter, with adjustments made as necessary.
A flow-splitting chamber was used for each test concentration (treatment) to promote mixing of the test solution and to allocate equally the test solution among the four replicate test chambers.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Obtained from Oregon State University's Sinnhuber Aquatic Research Laboratory (SARL, Corvallis, OR, USA).

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS: No data

POST-HATCH FEEDING
- Start date: Feeding began on day 5 of the test.
- Type/source of feed: The hatched organism were fed paramecium slurry ( obtained from Oregon State University's Sinnhuber Aquatic Research Laboratory [ SARL, Corvallis, OR, USA]) and a small scoop of Larval AP100/ Artificial Plankton Rotifer Powder (L/A powder) (obtained from Ziegler Bros., Inc. [Gardeners, PA, USA] and Aquatic Eco-system, Inc. [Apopka, FL, USA], respectively). On day 15, the feeding of the paramecium slurry was stopped and organism only received the brine shrimp and the L/A powder. As the test progressed, the amount of brine shrimp fed to all chambers was increased and the feeding of L/A powder was stopped when the organisms' stomach were red with brine shrimps. As the test progressed, the amount of brine shrimp fed to all chambers was increased and the feed of L/A powder was stopped when the oragnisms' stomachs were red with brine shrimp.

- Amount given: 3 mL paramecium slurry and a small scoop (approximately 4.0 mg) of Larval AP100/ Artificial Plankton Rotifer Powder (L/A powder).On day 10 of the test, the amount of paramecium slurry was reduced to 1.5 mL and 0.15 mL of live brine shrimps (Artemia salina) nauplii was added along with the L/A powder.If very little or no brine shrimp was left, the feeding rate was increased 0.05 mL per feeding, accordingly.
- Frequency of feeding: Fed twice a day.
Test type:
not specified
Water media type:
freshwater
Total exposure duration:
30 d
Remarks on exposure duration:
Post-hatching(35 days total)
Post exposure observation period:
Not reported
Hardness:
95±3.9 mg/L as CaCO3
Test temperature:
25±2°c
pH:
7.5-8.0
Dissolved oxygen:
All measured Dissolved oxygen concentration were above 60% of saturation at the average test temperature(7.9 mg/L at 27°C)
Salinity:
Not applicable
Nominal and measured concentrations:
0 (control), 1000, 2000, 4000, 8000 and 16000 µg Mn/L - Nominal
23.09, 513.38, 1034.96, 2102.96, 4496.89 and 9334.70 µg/L - Average Measured Dissolved Mn Concentration
Details on test conditions:
TEST SYSTEM
- Embryo cups ( fill volume): 23-28 volume turnovers per day
- Test vessel:1-L rectangular glass chambers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Holds approximately 400-600 mL of testing solution and were divided into two compartments using nylon mesh.
- Type of flow-through : A continuous- flow proportional diluter with a dilution factor of 0.5 was used in the test
- Renewal rate of test solution (frequency/flow rate): The flow rate of each test chamber was approximately 7.5 mL/ min providing approximately 18 to 27 volume exchanges per replicate chamber per day.
- No. of fertilized eggs/embryos per vessel: 25 D.rerio eggs . A total of 100 organism will be tested in four replicatesfor each treatment.
- No. of vessels per concentration (replicates):4
- No. of vessels per control (replicates):4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution and control water used for this study was laboratory blended water ( well water blended with reverse osmosis-treated water with a targeted hardness of 80-120 mg/L as CaCO3). Laboratory blended water was heated to test temperature (25±2°c) prior to delivery to the test system.
- Total organic carbon: 3.1mg/ L. DOC = dissolved organic carbon. Measured via combustion (EPA 415.1; CH2M HILL, Corvallis, OR).
- Chlorine:37.3 mg/L .Measured via ICP Atomic Emission Spectroscopy (EPA 200.7; CH2M HILL, Corvallis, OR)
- Alkalinity:116±5.7 mg/L as CaCO3
- Ca/mg ratio: 6.21
- Conductivity:315±13.3 µS/ cm00
- Intervals of water quality measurement:The hardness and alkalinity were measured weekly during the test in the control/ dilution water and was measured in the dilution water at initiation. Temperature, dissolved oxygen (DO), pH, and conductivity were measured in each concentration at test initiation and daily thereafter.

OTHER TEST CONDITIONS
- Photoperiod:Lighting was controlled to provide a 16:8 hour light; dark cycle.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations of live and dead eggs were conducted on a daily basis from initiation to thinning(48 hours), and then to full hatch (day 10).
Following hatching, mortalities were recorded daily. At test termination, the fish in each test chamber were counted, exained for gross observable abnormalities. The fish from each replicate were then placed into a pre- weighed pan, dried at 105°c for over 12 hours, and then re-weighed to the nearest 0.01 mg to obtain a dry weight.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: Concentrations were selected based on findings from a previous fathead minnow (Pimephales promelas) manganese test (ENSR 1996).

POST-HATCH DETAILS
- No. of hatched eggs (alevins)/treatment released to the test chamber: The number of organisms in each cup was thinned to 25 viable embryos.
- Release of alevins from incubation cups to test chamber on day no: 10

FERTILIZATION SUCCESS STUDY
- Number of eggs used: 35 freshly fertilised eggs used
- Removal of eggs to check the embryonic development on day no: Observations of live and dead eggs were conducted on a daily basis from initiation to thinning (48 hours), and then to full hatch (day 10).
Reference substance (positive control):
not specified
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
4 496.89 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Basis for effect:
other: Survival
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
9 334.7 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Basis for effect:
other: Survival
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
4 629.1 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
other: Reduce organism survival by 10%
Remarks on result:
other: 95% CL
Duration:
35 d
Dose descriptor:
other: EC20
Effect conc.:
5 121.1 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
dissolved
Basis for effect:
other: Reduce the organism survival by 20%
Reported statistics and error estimates:
All statistical analyses were performed using the mean dissolved Mn concentrations. Difference in survival and growth at test termination were evaluated using statistical computer packages (TRAP, U.S. EPA and CETIS, Tidepool Scientific Software [McKineleyville, CA, USA]). If the data met the assumptions of normality and homogeneity, the NOEC and LOEC were estimate using an analysis of variance (Dunnett's Multiple Comparison or Bonferroni Adjusted t) to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. The EC10 and EC20 values were estimated using a piecewise linear regression. Exposure concentrations were log- transformed before determination of EC10 and EC20 values.

Table 6. Median Day to Hatch

 

Average Measured Dissolved Concentration (µg/L)

Median Day to Hatch by Replicate

Median Day to Hatch by Treatment (±SD1)

23.09

A=4.0, B=4.6, C=4.1, D4.5

4.3±0.3

513.38

A=4.2 B=4.2 C=4.4 D4.7

4.4±0.3

1034.96

A=4.9 B=3.4 C=4.3 D4.5

4.3±0.7

2102.96

A=4.6 B=4.8 C=4.3 D3.7

4.3±0.5

4496.89

A=4.2 B=4.3 C=4.2 D4.4

4.3±0.1

9334.70

A=4.4 B=3.8, C=4.0 D4.0

4.0±0.3

1SU = Standard Units

 

Table 7. Test Survival Data

 

Average Measured Dissolved Mn Concentration

(µg/L)

Number of Organism (% Survival)

Average % Survival(±SD1)

Sig. Less than the Control2

Rep A

Rep B

Rep C

Rep D

23.09

21(100)

22(100)

22(88)

23(96)

96±5.7

-

513.38

21(100)

21(91)

21(88)

25(100)

95±6.3

No

1034.96

22(96)

27(100)

19(95)

24(100)

98±2.7

No

2102.96

21(91)

24(96)

23(100)

20(91)

95±4.3

No

4496.89

25(93)

20(87)

20(83)

21(95)

90±5.5

No

9334.70

3(14)

8(33)

5(19)

3(13)

20±9.4

Yes

1 SD = Standard Deviation

2 Significantly less than the control (p = 0.05) using Dunnett’s Multiple Comparison.

Note. The 35-d median lethal concentration (LC50) (with 95% confidence intervals) values was 6955.90 (6588.30 – 7344.0)µg/L dissolved Mn.

 

Table 8. Mean Dry Weight

 

Average Measured Dissolved Mn Concentration

(µg/L)

Number of Organism (% Survival)

Average % Survival(±SD1)

Sig. Less than the Control2

Rep A

Rep B

Rep C

Rep D

23.09

1.66

1.70

1.65

1.62

1.66±0.04

-

513.38

1.45

1.36

1.76

1.57

1.54±0.17

No

1034.96

1.73

1.24

1.68

1.26

1.48±0.26

No

2102.96

1.65

1.22

1.42

1.51

1.45±0.18

No

4496.89

1.41

1.69

0.75

1.75

1.40±0.46

No

9334.70

10.27

3.56

8.56

9.45

7.93±3.01

N/A3

 

1SD = Standard Deviation

2Significantly less than the control (p = 0.05) using Dunnett’s Multiple Comparison.

3. Concentration was not used in statistical analysis ( hypothesis testing) because a survival effect was observed at this concentration .

 

Table 9. Mean Dry Weight Per Original Organism (Biomass)

  

Average Measured Dissolved Mn Concentration

(µg/L)

Number of Organism (% Survival)

Average % Survival(±SD1)

Sig. Less than the Control2

Rep A

Rep B

Rep C

Rep D

23.09

1.66

1.70

1.45

1.55

1.59±0.11

-

513.38

1.45

1.24

1.54

1.57

1.45±0.15

No

1034.96

1.65

1.24

1.60

1.26

1.44±0.22

No

2102.96

1.50

1.17

1.42

1.38

1.37±0.14

No

4496.89

1.31

1.47

0.63

1.67

1.27±0.45

No

9334.70

1.47

1.19

1.63

1.18

1.37±0.22

No

1SD = Standard Deviation

2Significantly less than the control (p = 0.05) using Dunnett’s Multiple Comparison

 

Table 10. Summary of Test Statistics (µg Mn/ L)1

 

Evaluation

Survival

Dry weight

Biomass

NOEC

4496.89

4496.89

9334.7

LOEC

9334.70

>4496.89

>9334.7

EC10(95% CI)

4629.10(4299.80-4985.80

-

-

EC20( 95% CI)

5121.10(4788.80-5478.80)

-

-

1 Note. No Observed Effect Concentration. LOEC = Lowest Observed Effect Concentration. EC10= Effective Concentration for 10% reduction in endpoint relative to control performance. EC20 = Effective Concentration for 20% reduction in endpoint relative to control performance. CI= Confidence intervals.

Validity criteria fulfilled:
yes
Conclusions:
There was no significant effects on organism dry weight and biomass (dry weight per original organism) at any of the concentrations tested. The lowest NOEC was 4.5 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.

Data source

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

Materials and methods

Test material

Reference
Name:
Unnamed
Type:
Constituent

Results and discussion

Effect concentrationsopen allclose all
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
4 496.89 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: Dissolved
Basis for effect:
other: Survival
Duration:
35 d
Dose descriptor:
LOEC
Effect conc.:
9 334.7 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: Dissolved
Basis for effect:
other: Survival
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
4 629.1 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Dissolved
Basis for effect:
other: Reduced organism survival by 10 %
Duration:
35 d
Dose descriptor:
other: EC20
Effect conc.:
5 121.1 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: Dissolved
Basis for effect:
other: Reduced organism survival by 20 %

Applicant's summary and conclusion