Zinc oxide

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline followed but result is unbound NOEC and non-measured data

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Measurements of zinc concentrations in test solutions
- Sampling method: Test water acidified
- Sample storage conditions before analysis: 4 degree celsius

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Protocol for nanoparticle dispersion developed by National Physics Laboratory. Briefly nanoparticles were made into a paste using a few drops of DI water and then 10 drops of water was added to the paste and slowly stirred with a glass rod. After this the remaing water is added and the solution is sonicated twice for 10 sec

- Controls: RO water

- Evidence of undissolved material (e.g. precipitate, surface film, etc):End of study a fine doposition of material in exposure tanks from 60-540µg/L ZnO NP. Much of the depositied material was either coating the natural biofilm in the tanks or was bound to dead Artemia and faeces, suggesting that much of the material added to the tank may have been rapidely depositied.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test was initiated with Danio rerio(WIK strain) embryos from mass spawining stocks held at Cefas, Weymouth. Information on the orginal strain can be supplied by AstraZeneca where stocks originate from (8th Oct 2010). Fish were fed newly hatched Artemia spp. supplemented with commercial flake food daily. These fish supplied an F1 generation at Cefas from individual pairings. These F1 fish were then maintained in identical conditions to the parents prior to provision of embryos.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

35 d

Post exposure observation period:

Larval total length, larval dry weight, larval survival and delay in hatching

Hardness:

General water hardness - 260+/- 20 mg/L
Carbonate Hardness (KH) - 190 +10 mg/L

Test temperature:

25+/-2 °C

pH:

8.107-8.192

Dissolved oxygen:

Minimum 80% of the air saturated value

Nominal and measured concentrations:

540, 180, 60, 20 and 7 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 10L glass aquaria modified with outflow at 8L capacity and 10L glass aquaria modified modified with 4 mid-volume capacity
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass, 10L, unknown and 8L
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): water inflow rate to mixing vessel - 200 ml/min and dosing inflow rate to mixing vessel - 2.5 ml/min
- No. of organisms per vessel: 30
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: N7A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Declorinated water from supply to the laboratory.
- Total organic carbon: <1 mg/L
- Particulate matter: <3 mg/L

OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h dark
- Light intensity: 1500-1900 lux

TEST CONCENTRATIONS
- Test concentrations:540, 180, 60, 20 and 7 µg/L

Reference substance (positive control):

yes

Remarks:

yes (Micro-scale ZnO reference substance and ZnCl reference substance are used to compare against nano-scale ZnO.)

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 540 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: larval total length

Remarks on result:

other: duration in days post-hatching

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 540 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: larval dry weight

Remarks on result:

other: duration in days post hatching

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 540 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: duration in days post hatching

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 60 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: delay in hatching

Remarks on result:

other: data based on cumulative number of larvae hatching on day 4 and 5

Details on results:

- Observations on body length and weight: No dose-dependent effect on body-length or weight. 540µg/L had an effect on body length but not significant.
- Other biological observations: Post-hatch survival - no dose-dependent effect
- Mortality of control: <70% of post hatch survival of control individuals after 32 days
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Reported statistics and error estimates:

Mean and stardard deviation (SD) were calculated using Excel 2007. Survival and development data were analysed using Stata/IC software v11.2 for Windows. REsponses of the form r/n were analysed as binomal responses within a generalized linear model (GLM), using number of eggs or number hatched as denominators. Length and weight were analysesd by anova and checked with Kruskal and Wallis anova of ranks when Bartlett´s test indicated uneqqual variance. When anove indicated some difference of means the pattern was examined using Scheffe post-hoc test. NOEC and LOEC values were derived from Wald tests on model parameters. Distributions of values and patterns of response were examined graphically.

Over the exposure period, post hatch survival of control individuals was <70% (at 32dph). 

 A summary of the observations on zebrafish hatching success, growth and survival of individuals exposed to ZnO nanoparticles (NM-110 = Z-COTE), ZnO bulk material (NM-113) and ionic zinc (Zn⁺⁺)

 

Nominal test concentration   (µg /L)	Measured test concentration as mean ± SD (µg ZnO/L)	Larvae alive on day 35	Mean % survival at hatching and day 32 post hatch		Standard Length at 32d post hatch	Dry weight at 32d post hatch^3
			Hatching^1 (mean ± SD)	32d ph^2 (mean ± SD)	(mm)  (mean ± SD)	mg (mean ± SD)
Control	N/A	38	62 (1.9)	62 (5.3)	17.4 (3.0)	13.9 (6.8)
Zn^++	N/A	30	58 (8.8)	44 (8.8)	14.5 (4.8)	9.9 (8.1)
NM-113 180	N/A	29	70 (15.6)	37 (11.5)	15.9 (4.6)	10.5 (8.1)
NM-110 540	N/A	28	65 (5.8)	83 (9.9)	17.3 (3.6)	11.2 (5.9)
NM-110 180	N/A	33	53 (2.7)	68 (11.1)	18.6 (3.3)	15.1 (7.1)
NM-110 60	N/A	29	64 (3.2)	38 (14.2)	17.2 (4.1)	12.3 (8.1)
NM-110 20	N/A	38	59 (7.4)	54 (19.8)	18.0 (2.8)	17.6 (8.2)
NM-110 7	N/A	56	57 (8.2)	43 (7.9)	16.2 (3.8)	13.5 (7.4)

¹survival observations calculated from initial stocking numbers

²survival observations calculated from hatched individuals

³means based on censored data (Annex VII-Growth Data15.1)

 

 

 

Summary of life cycle effects in Zebrafish exposed to zinc oxide nanoparticles expressed as LOEC and NOEC concentrations based on nominal values.

 

End point	DPH^a	NOEC^b(µg/L)	LOEC^c(µg/L)
Larval total length	32	≥540	>540
Larval dry weight	32	≥540	>540
Larval survival	32	≥540	>540
Delay in hatching	<7	≥60^d	180^d

^aDPH = days post hatch

^bNOEC = No-observed-effect concentration

^cLOEC = Lowest –observed-effect concentration

^dbased on cumulative number of larvae hatching on day 4 and day 5

Validity criteria fulfilled:

yes

Conclusions:

Tests done according to standard protocol but giving an unbound NOEC

Executive summary:

_{Danio rerio embroys were exposed to a various of doses of ZnO nanoparticles (Z-COTE). The endpoints measured were}

_{Embryo survival, Hatching success, Delay (if any) in hatching, Larval survival (32dph), Larval weight at end of study (32dph) and standard larval length at the end of the study}

The results from the biological data are discussed for time to hatching, developmental and survival. There appears to be a slight delay in the hatching of larvae exposed to >180 µg/L nZnO, however, this may be the result of a data bias from one replicate tank and repetition of the study is required to confirm this finding. As no clear dose-dependent effect on growth or survival was observed with respect to exposure to ZnO nanoparticles in the range tested, both NOEC and LOEC values have been defined as > 540 µg/L. Data from the additional bulk ZnO and Zn⁺⁺positive control treatments suggest that at similar concentrations of 180 µg/L ionic zinc and bulk ZnO have an inhibitory effect on growth compared to nZnO. The poor growth rates, high mortality at the transition from yolk-sac stage to independent feeding and a notable number of undersized fish at the end of the study all indicate that the larvae were either not feeding properly or the dietary regime was sub-optimal. Following the transition to an Artemia only diet there was always prey items present in the water column of the exposure tanks and at termination on day 35, all individuals measured under the microscope were observed to have food in the gut.

 

Poor survival during the embryonic stage and immediately post hatching suggests that the batch of eggs used in this study were of poor quality. Additional handling and staging of individual eggs at the start of the study may also have attributed to the poor survival. Despite the low survival, no clear dose related effects following exposure to zinc oxide nanoparticles in the concentration range tested were observed were apparent.

Description of key information

Per the 64^th meeting of the ECHA Member State Committee (MSC), one of the three environmental toxicity study requests (fish testing) was removed from the draft in advance of the meeting, based on PfAs received, which MSC agreed to since fish were proven to be less sensitive to Zn²⁺ ions.

 

Nonetheless, for freshwater, the comparison of the chronic ecotoxicity data obtained within the same studies for the same species/endpoints after exposing the organisms to either the soluble Zn2+ion or the ZnO-NP form show that there is generally no difference in toxicity between the two Zn-forms.

The general observation of similar toxicity by the Zn-ion and the ZnO-NP is most relevant for the discussion on the aquatic PNEC developed for zinc substances; it can be concluded that the available data show that the general Zn-ion based PNEC for freshwater is relevant for the nano-ZnO, too.

Additional information

Nano ZnO chronic toxicity

The present review focused notably on these chronic data, which are considered to be of major relevancy for the risk assessment and the related PNECs. Like for the acute toxicity, the data are presented in the attached tables as normalized values with the ecotoxicity value observed for the soluble zinc salts (Zn2+) as reference (ECx = 100%). The ratio ECx Zn2+/ECx nano ZnO is provided. The detail of the studies is available in the relevant IUCLID sections.

The freshwater chronic dataset now covers all main taxonomic groups, with 2 invertebrates (mollusc and crustacean), 2 algae, 1 fish species and 1 aquatic plant species. In addition, data on 1 sediment crustacean is also included. The full set of EC10/NOEC values are presented in the attached tables.

The marine chronic dataset covers all main taxonomic groups, with 1 invertebrate (clam), 2 algae, 1 fish species.

For freshwater, the comparison of the chronic ecotoxicity data obtained within the same studies for the same species/endpoints after exposing the organisms to either the soluble Zn2+ion or the ZnO-NP form show that there is generally no difference in toxicity between the two Zn-forms.

The general observation of similar toxicity by the Zn-ion and the ZnO-NP is most relevant for the discussion on the aquatic PNEC developed for zinc substances; it can be concluded that the available data show that the general Zn-ion based PNEC for freshwater is relevant for the nano-ZnO, too.