Reaction mass of copper oxide and manganese dioxide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: ISO 10706

Version / remarks:

ISO 10706:2000 — water quality — determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea). ISO/TC 147/SC5 — water qual. methods; 2000 [Switzerland, 17 pp.].

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Principles of method if other than guideline:

The study carried out a comparative evaluation of acute and chronic toxicity of CuO nanoparticles and bulk metal oxide using Daphnia magna and Vibrio fischeri as test specimen. The chronic toxicity study on bulk CuO and Daphnia magna acc. to ISO 10706:2000, Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea) was reported here. Results of the acute toxicity test acc. to ISO 6341 (Determination of the inhibition of the mobility of Daphnia magna Straus) were only reported in the result section.

GLP compliance:

not specified

Remarks:

Existing studies (non-GLP): acc. to REACH Annex XI section 1.1, the use of existing data may be considered as a valid justification that testing is scientifically unjustified if the conditions stipulated are met.

Specific details on test material used for the study:

- purchased from Vetec, Rio de Janeiro, Brazil
- purity: 99%

Analytical monitoring:

yes

Remarks:

free fraction of copper ions in copper oxide suspension of 2000 mg/L

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Stock solutions of CuO bulk particles were prepared at a concentration of 4000 mg/L in ultrapure water. Stock uspensions were stored in the dark at 4 °C.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: D. magna Straus, 1820, Cladocera, Crustacea
- Culturing method: according to ISO 6341 and DIN 38412-30 protocols
- Culture medium: according to the guideline, M4 culture medium
- Feeding during test: yes
- Food type: algae, Scenedesmus subspicatus
- Amount: 5 × 10^6 cells per individual organism

ACCLIMATION
- Acclimation conditions (same as test or not): yes
- Type of food: algae, Scenedesmus subspicatus; green algae S. subspicatus were cultivated in CHU growth medium

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

not reported

Test temperature:

20 ± 2 °C

pH:

not reported

Dissolved oxygen:

not reported

Nominal and measured concentrations:

Nominal test concentration range: 7.8, 15.6, 31.2, 62.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker, 50 mL
- Test suspension volume: 25 mL
- Renewal rate of test solution: every 48 hours
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10 (M4 medium)
- No. of vessels per positive control (replicates): 10 (copper sulfate)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 culture medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: not reported

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h
- Light intensity: diffuse luminosity

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Reproduction/fecundity: daily, 21 days
- Growth: daily, 21 days
- Longevity (surviving animals): daily, 21 days

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline:
The four test dilutions used were defined according to results observed in the acute toxicity test and followed a geometric progression with a ratio of 2. The lowest test concentration for the definite test started from the concentration that had no effect on the exposed organisms over 48 h in the acute toxicity test.

- Range finding study: Concentration range was based on acute toxicity study for Daphnia magna.
- Test concentrations: range 3.6 – 2000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

copper sulfate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

15.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

31.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

7.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth

Remarks:

parental daphnids

Remarks on result:

other: see overall remarks

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

< 7.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth

Remarks:

parental daphnids

Remarks on result:

other: see overall remarks

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 62.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

mortality

Remarks:

parental daphnids

Details on results:

For a summary of results, please see any other information on results.

- Behavioural abnormalities: Significant increase of the number of neonates at the two highest copper oxide concentrations and the positive control compared to the blank control. The two treatments with the highest copper oxide concentrations were the only treatments significantly different to the control (please see comment in overall remarks). The derived 21d-NOEC was 15.6 mg CuO/L, the derived 21d-LOEC was 31.2 mg CuO/L.
- Observations on body length and weight: Average growth of neonates significantly decreased with increasing test concentration (no clear dose-response relationship, please see comment in overall remarks). Significant effects could be observed at the lowest concentration: therefore, the 21d-LOEC was 7.8 mg CuO/L (please see comment in overall remarks).
- Other biological observations: morphological changes: shortening of the apical spine
- Mortality of control: no
- Derived effect concentrations of the acute immobility test with Daphnia magna were: 48h-EC50 (immobility, Daphnia magna) = 223.6 ± 9.2 mg CuO/L
- The measured free faction of copper ions (Cu 2+) for the copper oxide stock solution of 2000 mg/L was 0.37 ± 0.08 mg Cu/L.

Results with reference substance (positive control):

Chronic exposure to copper sulfate did not have a significant effect on reproduction of Daphnia magna, i.e. no adverse effect was observed at 0.06 mg CuSO4/L. The 48h-EC50 (Daphnia magna, immobility) for copper sulfate was 0.10 ± 0.25 mg/L.

Reported statistics and error estimates:

Results were analyzed statistically using one-way ANOVA according to EPA 821-R-02-013. The level of significance was set to p < 0.05.

Effect concentrations for acute toxicity during 48 hours and long-term effects on reproduction, growth and survivla of Daphnia magna exposed to CuO nano and CuO bulk particles during 21 days. Results represent the mean (n=10) ± SD.

	Acute	Chronic
	EC50 (mg /L)	Concentrations (mg/L)	Reproduction: No. of neonates/brood	Growth: Length (mm)	No. of survivors
CuO nano		Control	18 ± 2	5.02 ± 0.11	1 ± 0
		0.9	17 ± 2	4.49 ±0.36^+a	1 ± 0
	22 ± 0.70	1.9	18 ± 3	4.42 ± 0.34^+	1 ± 0
		3.9	18 ±7^b	4.00 ± 0.20^+	1 ± 0
		7.8	21 ±3^⁎,a	4.39 ± 0.37^+	1 ± 0
CuO bulk		Control	18 ± 2	5.02 ± 0.11	1 ± 0
		7.8	19 ± 2	4.61 ±0.44^+a	1 ± 0
	223.6 ± 9.2	15.6	18 ±3^b	4.36 ± 0.39^+	0.9 ± 0.31
		31.2	20 ±2^+a	4.23 ± 0.41^+	1 ± 1
		62.5	19 ±2^+a	4.44 ± 0.46^+	0.9 ± 0.31
CuSO4		Control	18 ± 2	5.02 ± 0.11	1 ± 0
	0.10 ± 0.25	0.06	21 ± 4^+	4.27 ± 0.44 ^+	1 ± 0

+ Values significantly different from control (p < 0.05).

^a LOEC — low observed effect concentration

^b NOEC — no observed effect concentration

Validity criteria fulfilled:

yes

Remarks:

OECD 211

Conclusions:

The derived effect concentrations were: 21d-NOEC (reproduction, Daphnia magna) = 15.6 mg CuO/L (nominal), 21d-LOEC (reproduction, Daphnia magna) = 31.2 mg CuO/L (nominal), 21d-NOEC (survival, Daphnia magna) >= 62.5 mg CuO/L (nominal); measured free faction of copper ions (Cu 2+) for copper oxide stock solution of 2000 mg/L was 0.37 ± 0.08 mg Cu/L.

Executive summary:

The study carried out a comparative evaluation of acute and chronic toxicity of CuO nanoparticles and bulk form using Daphnia magna and Vibrio fischeri. The chronic toxicity study on Daphnia magna was conducted according to ISO 10706 (similar to OECD Guideline 211), and the range of test concentrations were based on results of the acute toxicity test performed beforehand. The nominal test concentrations were 7.8, 15.6, 31.2, 62.5 mg/L copper oxide. A blank and positive control (copper sulfate, 100% bioavailable copper) were run in parallel. Analytical measurements were only performed on the bioavailable copper fraction of the stock suspension (2000 mg/L).

After 21 days of exposure, onset of birth of offspring was similar for all treatment groups including copper sulfate, indicating that copper oxide had no effect on start of reproduction. The measured effects on reproduction did not follow a typical dose-response relationship. There was a significant increase of the number of neonates relative to the blank control for copper oxide at 31.2 mg/L and copper sulfate at 0.06 mg/L. The determined 21d-NOEC for copper oxide on reproduction of Daphnia magna was 15.6 mg/L (based on increased number of offspring).

Although no significant effects on the survival of neonates (longevity) were observed during 21 days of exposure, some morphological changes were found at the end of the test, i.e. shortening of the apical spine. Effects on parental growth were observed at all test concentrations including the positive control, but without a dose-dependent pattern. The effect on growth was related to feed shortage caused by the negative effect of free copper on algae growth, which was used as feed. The effect was therefore not used as reliable endpoint for the inherent toxicity of copper oxide on Daphnia magna. 

Despite shortcomings in reporting, the results of the study were considered reliable and were therefore used to evaluate adverse effects on aquatic organisms. Results of the acute toxicity test reported in Heinlaan et al. 2008 (see section 6.1.3) were comparable to effect concentrations reported here, thereby supporting the reliability of results reported by Rossetto et al. 2012.

Description of key information

21d-NOEC (Daphnia magna, reproduction) = 69 mg/L (nominal, total fraction, submission substance), corresponding to 0.028 mg/L dissolved copper (measured / extrapolated) (ISO 6341)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.028 mg/L

Additional information

A reliable key study was available for the hazard assessment of chronic toxicity to aquatic invertebrates for the submission substance reaction mass of copper oxide and manganese dioxide. The available key study was performed with copper oxide. Copper oxide was considered the hazardous constituent of the submission substance with regard to the aquatic environment. No study was available for chronic toxicity of manganese dioxide in aquatic invertebrates. 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 oxide was considered protective (see also attached read-across report, section 13).

The key study on constituent copper oxide investigated chronic toxicity to Daphnia magna according to ISO 10706 (similar to OECD Guideline 211) (RL2, 2014). Four nominal test concentrations were used. A blank and positive control (copper sulfate, 100% bioavailable copper) were run in parallel. After 21 days of exposure, onset of birth of offspring was similar for all treatment groups including copper sulfate, which indicated that copper oxide had no effect on start of reproduction. The measured effects on reproduction did not follow a typical dose-response relationship. There was a significant increase of the number of neonates relative to the blank control. The observed effect was interpreted as the organisms' attempt to preserve the species under stressful conditions, which had been reported previously. The effect was also observed in the treatment group with copper sulfate. Although no significant effect on the survival of neonates (longevity) was observed during 21 days of exposure, some morphological changes were found at the end of the test, i.e. shortening of the apical spine. Effects on parental growth were observed at all test concentrations including the positive control, but without a dose-dependent pattern. The effect on growth was related to feed shortage caused by the negative effect of free copper on algae growth, which was used as feed. The effect was therefore not used as reliable endpoint for the inherent toxicity on Daphnia magna. In conclusion, the nominal 21d-NOEC on reproduction of Daphnia magna was 69 mg/L (based on increased number of offspring; value recalculated based on the ratio of MnO2 and CuO in the reaction mass), corresponding to 0.028 mg/L dissolved copper (value extrapolated based on measured dissolved copper fraction of 0.18% for nominal CuO concentration, see section 6.1.5, Aruoja et al. 2009).