Reaction mass of copper oxide and manganese dioxide

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Version / remarks:

The study fowllowed: U.S. EPA, 1972, Proposed recommended bioassay procedure for egg and fry stages of freshwater fish. U.S. Environmental Protection Agency, National Water Quality Laboratory, Duluth, MN; OPPTS 850.1400 (1996) was inter alia based on the proposal

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Principles of method if other than guideline:

The study investigated effects of exposure to different heavy metals on six freshwater fish species (rainbow trout, lake trout, channel catfish, white sucker, bluegill, northern pike, walleye, brook trout). To this end, adverse effects of copper, cadmium, chromium and lead on eggs and fry were examined in soft and hard water. Results for copper on the most sensitive fish species were reported here (brook trout). The study followed the U.S. EPA proposed recommended bioassay procedure for egg and fry stages of freshwater fish (later as part of EPA OPPTS 850.1400).

GLP compliance:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity test date: analytical grade

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations, including control
- Sample size per concentration: 7 replicates (replicates of analytical measurements of test concentrations)
- Sampling method: Unfiltered water samples were collected in polyethylene bottles. Concentrated nitric acid was added to each sample (1% by volume) before storage.
- Sample storage: Samples were stored at room temperature prior analysis for 2-3 weeks.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Stock solution was prepared with distilled water at a concentration of 1000 mg/L. The test solution was delivered to diluters from a Mariotte bottle by a volumetric delivery system. The diluent water was obtained from a 400 ft. bedrock well.

Test organisms (species):

Salvelinus fontinalis

Details on test organisms:

TEST ORGANISM
- Common name: brook trout
- Source: Wareham, Mass., USA
- Age of eggs at test start: 0-1 day
- Incubation time: 35 days

POST-HATCH FEEDING
- Type/source of feed: Oregon moist trout starter, four times per day

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

60 d

Hardness:

hard water: 187.0 ± 22.0 mg/L as CaCO3
soft water: 37.5 ± 7.3 mg/L as CaCO3

Test temperature:

10 ± 1°C

pH:

hard water: 6.7-7.1
soft water: 6.6-7.1

Dissolved oxygen:

hard water: 11.0 ± 1.2 mg O2/L
soft water: 10.0 ± 0.7 mg O2/L

Nominal and measured concentrations:

Nominal (hard/soft): 100, 50, 25, 12, 6, 3 µg/L
Measured:
Soft water (µg/L):
95 ± 7.4
51 ± 5.1
27 ± 3.2
13 ± 2.1
7 ± 1.6
5 ± 1.2
Hard water (µg/L):
74 ± 9.4
49 ± 7.0
21 ± 5.1
13 ± 3.6
8 ± 2.3
5 ± 1.6

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria
- Size, fill volume: 35 x 30 x 30 cm, 20 cm water depth; two growth chambers were set in one aquarium (25 x 30 x 12.5 cm)
- Egg incubation cups: 4-oz., 2-inch 0D round glass jars, bottoms cut off and replaced with nylon screen; incubation cups oscillated in the test water by a rocker arm apparatus driven by a 2 rpm motor
- No. of fertilized eggs per aquarium: 200
- No of fry: 100 (number of fry was reduced to 100 per replicate or 50 per growth chamber); on day 30 fry were reduced to 50 per replicate (25 per growth chamber)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: obtained from a 400 ft. bedrock well (total hardness: 35 mg/L as CaCO3, for hard water bedrock water was passed through a water hardener).
- Metal background concentration: 3 µg/L copper
- Alkalinity: soft water: 30.0 ± 1.4 (mg/L as CaCO3); hard water: 177.0 ± 32.0 (mg/L as CaCO3)
- Water quality measurement: acidity, alkalinity, hardness, and pH of diluent water
- Maintenance: Aquaria were siphoned daily to remove fetal material, excess food and detritus. Aquaria were brushed when algal growth became excessive.

OTHER TEST CONDITIONS
- Light intensity: Eggs and fry were covered from all sources of light by black polyethylene curtains.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Hatchability of eggs: daily, determined after 30
- Survival: daily, determined after 30 and 60 days
- Growth: after 30 and 60 days

Reference substance (positive control):

no

Key result

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

5 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element (dissolved fraction)

Remarks:

copper

Basis for effect:

length

Remarks:

weight

Remarks on result:

other: soft water

Remarks:

37.5 ± 7.3 mg/L CaCO3; statistically reassessed acc. to OECD 210

Duration:

60 d

Dose descriptor:

LOEC

Effect conc.:

7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element (dissolved fraction)

Remarks:

copper

Basis for effect:

length

Remarks:

weight

Remarks on result:

other: soft water

Remarks:

37.5 ± 7.3 mg/L CaCO3; statistically reassessed acc. to OECD 210

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

copper

Basis for effect:

number hatched

Remarks on result:

other: soft water

Remarks:

37.5 ± 7.3 mg/L CaCO3; statistically reassessed acc. to OECD 210

Duration:

60 d

Dose descriptor:

LOEC

Effect conc.:

13 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element (total fraction)

Remarks:

copper

Basis for effect:

number hatched

Remarks on result:

other: soft water

Remarks:

37.5 ± 7.3 mg/L CaCO3; statistically reassessed acc. to OECD 210

Duration:

60 d

Dose descriptor:

NOEC

Effect conc.:

13 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element (dissolved fraction)

Remarks:

copper

Basis for effect:

mortality

Remarks on result:

other: soft water

Remarks:

37.5 ± 7.3 mg/L CaCO3; statistically reassessed acc. to OECD 210

Duration:

60 d

Dose descriptor:

LOEC

Effect conc.:

27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

element (dissolved fraction)

Remarks:

copper

Basis for effect:

mortality

Remarks on result:

other: soft water

Remarks:

37.5 ± 7.3 mg/L CaCO3; statistically reassessed acc. to OECD 210

Details on results:

For result table see any other information on results. Note, effect concentrations (NOEC / LOEC) were statistically reassessed for soft water acc. to OECD 210. Deviating levels of statistical significances in comparison to the publication were previously reported in the Voluntary risk assessment reports - Copper and Copper Compounds (2008) and used for the calculation of the species sensitivity distribution (SDD) for copper and copper compounds. See also other remarks, attachments, Note 2.

Soft water (37.5 ± 7.3 mg/L CaCO3):
In general, the observed effects followed a dose-response relationship. None of the fish eggs exposed to the highest concentration hatched successfully. For concentrations of 51, 27 and 13 µg/L copper, the percentage of hatching eggs were reduced significantly compared to the control. After 60 days of exposure, concentrations of 51 and 27 µg/L copper significantly reduced fry survival compared to the control. Concentrations >= 7 µg/L significantly reduced total length and wet weight of fry compared to the controls. Therefore, the derived effect concentrations were: LOEC (length/weight) = 7 µg/L; and NOEC (length/weight) = 5 µg/L.

- The MATC (maximal acceptable toxicant concentration) was estimated to be in the range of 3 and 5 µg/L by the authors.
- Survival of control: 100% day 0-30, 96-98% day 31-60
- Background concentration: The mean concentration of copper in the diluent water (control) had a measured concentration of 3 µg/L.

Hard water ( 187.0 ± 22 mg/L CaCO3):
In general, the observed effects followed a dose-response relationship. The percentage hatch was significantly reduced by exposure to the highest concentration of 74 µg/L compared to the controls. None of fry survived 30 day of exposure to 74 µg/L. Survival and total length were reduced after 30 days to 49 µg/L copper compared to the controls and other treatment groups.
None of the fry survived 60 days exposure to 49 µg/L copper. Survival in lower copper concentrations were comparable with controls. Total length and wet weight at the end of exposure were significantly reduced by exposure to 21, 13 and 8 µg/L, but survival was comparable with controls. Total length and wet weight at 5 µg/L copper were similar to controls.
The MATC (maximal acceptable toxicant concentration) was estimated to be in the range of 5 and 8 µg/L. Therefore, the corresponding LOEC was 8 µg/L and the NOEC was 5 µg/L.
- Survival of control: 100% day 0-30, 100% day 31-60 in replicate 2, 58% day 31-60 in replicate 1

Reported statistics and error estimates:

Soft water, NOEC / LOEC reassessment (March 2017) acc. to OECD 210 by ToxRat Standard Version 3.2.1 (released 02.11.2015):
Shapiro-Wilk´s Test and Levene´s Test (with Residuals) were applied to test for normal distribution and variance homogeneity, respectively. As none of the measured endpoints fullfilled the requirements for parametric testing, step-down Jonckheere-Terpstra test was applied (p<0.05).

Hard water:
Replicates were averaged and subjected to analysis of variance acc. to Steele and Torrie (1960)*. If treatment effects were indicated, statistical significance was analysed acc. to Duncan's Multiple Range Test (between treatment groups and control). Level of significance was set to p = 0.05.

*Steele, R.G.D. and J.H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York: 481 pp.

Soft water ( 37.5 ± 7.3 mg/L CaCO3)

Mean percentage hatch of eggs, mean survival, total length and wet weight of brook trout

Mean measured copper conc. (µg/l)			1-30 Days			31-60 Days
		Mean hatch (%)	Survival (%)	Mean total length (mm)	SD	Survival (%)	Mean total length (mm)	SD	Mean total wet wt.(mg)
95	A	0 *§	-	-	-	-	-	-	-
	B	0	-	-	-	-	-	-	-
51	A	5 *§	0 *	-	-	-	-	-	-
	B	2	2	18	1.5*	0	-	-
27	A	6 *§	10 *	19	1.8*	8*§	18	1.2 *§	80 *§
	B	4	6	18	1.9	6	17	3.8	96
13	A	43 *§	96	19	1.8*	96	22	7.1 *§	120 *§
	B	25	74	18	1.5	72	23	2.4	101
7	A	63	100	22	2	98	24	3 *§	126 *§
	B	72	98	21	1.7	98	24	4.2	127
5	A	72	100	22	2.3	98	26	3.3 *	144 *
	B	77	98	22	1.9	90	25	4	151
Control	A	79	100	22	2.2	96	28	5.3	192
	B	73	100	23	2.3	98	29	4.2	240

* = Value significantly lower than the controls (Duncan's Multiple Range, p<0.05), the asterics refers to significant differences among treatment groups taken the two groups together (A/B).

§ = statistical reassessment acc. to OECD 210 (ToxRat Standard Version 3.2.1): value significantly lower than the controls (Step-down Jonckheere-Terpstra, p<0.05), the section sign refers to significant differences acompared to the control taken the two groups together (A/B).

Hard water (187.0 ± 22 mg/L CaCO3)

Mean percentage hatch of eggs, mean survival, total length and wet weight of brook trout

Mean measured copper conc. (µg/L)		Mean hatch (%)	1-30 Days			31-60 Days
			Survival (%)	Mean total length (mm)	SD	Survival (%)	Mean total length (mm)	SD	Mean total wet wt. (mg)
74	A	19*	0*	-	-	-	-	-	-
	B	12	0	-	-	-	-	-	-
49	A	35	52*	17	1.6*	0*	-	-	-
	B	35	70	17	1	0	-	-	-
21	A	45	100	19	1.8	76	19	2.6*	47*
	B	44	90	19	1.7	58	19	1.6	48
13	A	48	100	20	1.2	76	22	1.3*	81*
	B	49	98	20	1.8	90	20	2.3	78
8	A	49	100	19	1.3	74	21	2.2*	79*
	B	53	100	20	1.4	86	19	2.1	75
5	A	45	100	20	1.5	68	24	2.2	110
	B	51	100	20	2.5	80	22	2	117
Control	A	56	100	20	1.7	58	27	1.8	117
	B	39	100	20	1.4	100	24	3.2	128

* = Value significantly lower than the controls (Duncan's Multiple Range P=0.05), the asterics refers to significant differences among treatment groups taken the two replicates together (A/B).

Validity criteria fulfilled:

not applicable

Conclusions:

The derived 60d-LOEC (Salvelinus fontinalis, growth) = 7 µg Cu/L (dissolved, measured); the corresponding 60d-NOEC (Salvelinus fontinalis, growth) = 5 µg Cu/L (dissolved, measured).

Executive summary:

The study investigated effects of exposure to different heavy metals on six freshwater fish species (rainbow trout, lake trout, channel catfish, white sucker, bluegill, northern pike, walleye, brook trout). To this end, adverse effects of copper, cadmium, chromium and lead on eggs and fry were examined in soft and hard water. Results for copper on the most sensitive fish species were reported here (brook trout). The study followed the U.S. EPA proposed recommended bioassay procedure for egg and fry stages of freshwater fish (later as part of EPA OPPTS 850.1400).

Measured effects were percentage hatching, survival and growth of fry in six freshwater fish species. The most sensitive endpoint was juvenile growth in soft water for brook trout. Effects in hard water (187.0 ± 22 mg/L CaCO3) were in general less pronounced than in soft water (37.5 ± 7.3 mg/L CaCO3). Observed effects followed a dose-response relationship. None of the fish eggs exposed to the highest concentration hatched successfully. For concentrations of 51, 27 and 13 µg/L copper, the percentage of hatching eggs were reduced significantly compared to the control. After 60 days of exposure, concentrations of 51 and 27 µg/L copper significantly reduced fry survival compared to the control. Concentrations >= 7 µg/L significantly reduced total length and wet weight of fry compared to the controls. Therefore, the derived effect concentrations were: LOEC (length/weight) = 7 µg/L; and NOEC (length/weight) = 5 µg/L.

The study was considered reliable and results were used to assess adverse effects of copper towards auqatic organisms.

Description of key information

60d-LOEC (brook trout, growth) = 0.29 mg/L (measured / extrapolated, total fraction, submission substance), corresponding to 7 µg Cu/L (dissolved, measured); 60d-NOEC (brook trout, growth) = 0.21 mg/L (measured / extrapolated, total fraction, submission substance), corresponding to 5 µg Cu/L (dissoved, measured, similar to US EPA OPPTS 850.1400)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

5 µg/L

Additional information

A reliable key study was available for the hazard assessment of chronic toxicity to fish for the submission substance reaction mass of copper oxide and manganese dioxide. The available key study was performed with dissolved copper. Dissolved copper was considered the hazardous element of the submission substance with regard to the aquatic environment. No study was available for chronic toxicity of manganese dioxide in fish. As the constituent manganese dioxide was of very low water solubility, and no toxicity of manganese dioxide was observed in any of the acute toxicity studies on aquatic organisms, using effect concentrations on the basis of copper was considered protective (see also attached read-across report, section 13).

The key study investigated effects of exposure to different heavy metals on six freshwater fish species (rainbow trout, lake trout, channel catfish, white sucker, bluegill, northern pike, walleye, brook trout) (RL2, 1976). The study followed the U.S. EPA proposed recommended bioassay procedure for egg and fry stages of freshwater fish (later as part of EPA OPPTS 850.1400). To this end, adverse effects of copper on eggs and fry were examined in soft and hard water. Results for copper on the most sensitive fish species were used for aquatic hazard assessment.

Measured effects were percentage hatching, survival and growth in six freshwater fish species. The most sensitive endpoint was juvenile growth in soft water for brook trout. Effects in hard water (187.0 ± 22 mg/L CaCO3) were in general less pronounced than in soft water (37.5 ± 7.3 mg/L CaCO3). Observed effects followed in general a dose-response relationship. Hatching and survival of offspring were not effected at lowest copper concentration, and growth was the most sensitive endpoint. Derived effect concentrations based on growth were: LOEC = 7 µg Cu/L and the NOEC = 5 µg Cu/L (dissolved copper, measured). The respective effect concentrations based on the total fraction of the submission substance were 0.29 and 0.21 mg/L, respectively (value recalculated based on the dissolved copper fraction acc. to OECD 29, i.e. 2.42%, see section 4.8).