Ozone

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Standard bioassay procedures (EPA 1975)

Version / remarks:

EPA (1975) Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. Ecol. Res. Ser. EPA-660/3-75-099, U.S. Environ. Prot. Agency, Natl. Environ. Res. Cent., Corvallis, Oreg.

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 2, 7, 29 µg/L Median lethal ozone concentrations were calculated and the resulting toxicity curve was determined by the Fisheries Bioassay Laboratory, Montana State University, using a trimmed Spearman-Karber computer program (Hamilton et al.1971).
- Sampling method: Sampling procedures were those outlined in Wedemeyer and Yasutake (1977).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The ozonated test solution was obtained by adding ozone to test water by a sparger-fed, countercurrent, 2-m water tower as the contact chamber (see Fig 1).
- Eluate: no
- Controls: Dilution water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Age at study initiation: juvenile
- Length at study initiation: 10-13 cm
- Feeding during test: yes, but only a maintenance ration

ACCLIMATION
- Acclimation period: at least 30 d before each test + ca 1 week adaptation in test throughs.
- Acclimation conditions : laboratory water supply in holding tank and in test throughs
- Type and amount of food: the Oregon Moist Pellet diet (OMP)
- Feeding frequency: daily feeding, but withheld for 24 h before moving fish to the 24-L test throughs

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 mo

Remarks on exposure duration:

partial chronic

Post exposure observation period:

-

Hardness:

total hardness: 20 mg/L (as CaC03)

Test temperature:

10°C

pH:

6.8

Dissolved oxygen:

11 mg/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Mean ± SD = 2.3 ± 0.2 µg/L and 5.0 ± 0.4 µg/L (measured)

Details on test conditions:

TEST SYSTEM:
The equipment shown schematically in Fig. 1 was evolved to minimize the well-known difficulties of achieving stable dissolved ozone levels.
Water flows for each trough were set at 800 mL/min, resulting in a 95% replacement time of 0.5 hand density and flow indices of 1.2 and 2.0, respectively, approximately the same as in the holding tanks.

For the partial chronic exposure tests, 35 fish were randomly distributed to duplicate troughs and allowed to adapt for about a week. Initial blood and tissue samples were obtained from 10 fish randomly removed from the six troughs before turning on the ozone generator. Additional five fish blood and tissue samples were taken at 2, 4, 6, 8, 10, and 12 wk. As in the acute exposures, to prevent wide fluctuations in ozone demand, and thus in exposure level, it was necessary to clean the troughs daily and remove mortalities as they occurred. Only a maintenance ration could be fed.

TEST MEDIUM / WATER PARAMETERS
Cl- : 2.6 mg/L; Fe3+: 0.2 mg/L; N03-N: 0.5 mg/L; turbidity, 3.1 JTU.

TEST concentrations OZONE:
Due to the thermodynamic instability of ozone and the unavailability of feedback control systems, it was not possible to achieve completely stable, graduated, ozone exposure levels over long time periods and only partial chronic (3-mo) tests could be carried out.

Reference substance (positive control):

no

Key result

Duration:

3 mo

Dose descriptor:

NOEC

Effect conc.:

2 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

3 mo

Dose descriptor:

LOEC

Effect conc.:

5 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

At the 2 µg/L chronic exposure level, there were no mortalities in either the test or control groups and the only physiological change that could be associated with ozone was a mild thrombocytosis in the test fish (Table 1). There was a tendency toward lymphocytosis and a normochronic, nomocytic anemia in both the control and test fish, as indicated by the differential blood cell counts and the gradual decline in hemoglobin and haematocrit (Table 1, 2). A mild but persistent hypoglycaemia was also evident. Since these abnormalities were also seen in the control groups, they were undoubtedly the result of inanition caused by the minimal feeding regimen required to control fluctuations in ozone demand and help stabilize exposure levels. This was substantiated by the approximately equal weight loss in both the experimental and control groups (Table 2). The gill histological changes that occurred at the 2 microg/L exposure level were minor and apparently did not compromise functions such as ionoregulation -as indicated by the fact that stable plasma Na+ and Cl levels were maintained (Table 2).
At the 5 µg/L chronic exposure level, experience revealed that the feeding level could be increased enough to maintain a moderate weight gain before an excessively fluctuating ozone demand was created. Growth was thus normal in the controls (Table 3). However, although food was available, feeding behavior in the ozone-exposed groups began to decrease noticeably after ~2 mo and a significant (P = 0.05) within-group weight loss, together with a mild hypoglycemia, occurred during the 3rd mo. A mild polycythemia with falling immature erythrocyte counts and lymphocytopenia were also significant (P = 0.05) within-group trends (Table 3, 4). These were presumably physiological compensations reflecting impaired gas exchange due to the gradually developing gill hypertrophy. Histologically, the gill lamellae in the 5 microg/L test groups exhibited varying degrees of epithelial damage. Hypertrophy was more extensive but generally was qualitatively similar to that occurring at the 2 microg/L ozone exposure level. The one exception was that after 3 mo, hypertrophy of occasional individual epithelial cells in the 5 microg/L test groups resulted in an irregular-appearing lamellar surface. This was not seen at the lower exposure level. Again, the degree of gill damage that occurred did not compromise ionoregulation, as indicated by the stable plasma Na+,Cl- levels that were maintained (Table 3). In general, as was true of the acute ozone toxicity results, the gill pathological changes were not pathognomonic, but typical of those seen following exposure to agents such as chlorine, ammonia, and formalin (Wedemeyer 1971; Smart 1976).

Results with reference substance (positive control):

not applicable

TABLE 1. Summary of hematological changes in 10-13-cm rainbow trout caused by 3-mo chronic ozone exposure at 2 µg/L in 10°C soft water. Values are for means of differential cell counts per 1000 cells (mean ± SD) 10 fish per group. * Significant within group difference from initial values (P = 0.05).

Blood cell type and exposure group	Value after exposure (mo)
	0	1	2	3
Erythrocytes,mature
control	955±14	930±21*	942±18	944±20
test	955±14	944±25	954±38	927±33*
Erythrocytes,immature
control	31±13	44±18	16±13*	23±17
test	31±13	29±14	11±6*	20±16
Lymphocytes
control	13±7	26±10*	27±14*	32±23*
test	13±7	25±12*	31±31*	46±31*
Thrombocytes
control	0.2±0.4	0.1±0.3	1.5±3.2	1.9±3.2
test	0.2±0.4	3.6±3.0*	12.0±13.3*	8.2±6.2*
Neutrophils,mature
control	0.2±0.4	0	0.1±0.3	0
test	0.2±0.4	0	0.2±0.7	0.1±0.2
Neutrophils,immature
control	0.2±0.4	0	0	0
test	0.2±0.4	0	0.1±0.2	0

 

TABLE 2. Summary of blood chemistry changes in 10-13cm rainbow trout caused by 3-mo chronic ozone exposure at 2 µg/L in 10°C soft water. Values are given as means (x ± SD) 20 fish per group. None of the test groups was significantly altered from its corresponding control group (P = 0.05). *Significant within group difference from initial values (P = 0.05).

Physiological parameter and exposure group	Value after exposure (mo)
	0	1	2	3
Plasma chloride (meq/L)
control	134±6	130±3	133±3	132±2
test	134±6	129±3	130±4	130±4
Hematocrit (%)
control	45±7	40±3	34±5*	33±3*
test	45±7	43±5	36±7*	36±6*
Hemoglobin (g/100mL)
control	8.9±0.7	7.6±0.6*	6.6±1.2*	6.1±1.0*
test	8.9±0.7	7.8±O.7*	6.7±1.1*	6.6±1.0*
Growth (fish wt, g)
control	29±9	28±8	21±10*	19±6*
test	29±9	24±8	22±9*	21±10*
Plasma glucose (mg/100mL)
control	131±22	67±6*	72±5*	66±13*
test	131±22	68±8*	74±10*	75±11*
Plasma sodium (meq/L)
control	144±5	150±4	151±3	145±5
test	144±5	149±4	146±3	151±4

TABLE 3. Summary of physiological changes in 10-13-cm rainbow trout caused by 3-mo chronic ozone exposure at 5 µg/L in 10°C soft water. Values given as means (mean ± SD) 20 fish per group.*Significant within group difference from initial values (P = 0.05).

Physiological parameter and exposure group	Value after exposure (mo)
	0	1	2	3
Plasma chloride (meq/L)
control	133±4	134±3	130±3	121±4*
test	133±4	134±2	130±5	134±4
Hematocrit (%)
control	47±3	42±3	54±3*	38±3*
test	47±3	45±4	50±4*	45±8
Hemoglobin (g/dL)
control	7.6±0.6	7.6±0.6	7.3±0.3	7.0±0.6*
test	7.6±0.6	7.4±0.8	8.1±0.5	7.4±0.7
Growth (fish wt, g)
control	20±5	25±4*	38±10*	42±5*
test	20±5	25±7	35±12*	28±11*
Plasma glucose (mg/dL)
control	92±11	90±6	105±21	90±16
test	92±11	90±8	93±11	81±18*
Plasma sodium (meq/L)
control	159±2	153±3	157±4	143±5*
test	159±2	154±3	152±3	152±S

 

TABLE 4. Summary of hematological changes in lO-13-cm rainbow trout caused by 3-mo chronic ozone exposure at 5 µg/L in 10°C soft water. Values are for means of differential cell counts per 1000 cells (mean ± SD) 10 fish per group. *Significant within group difference from initial values (P = 0.05).

Blood cell type and exposure group	Value after exposure (mo)
	0	1	2	3
Erythrocytes, mature
control	922±31	924±22	864±33	921±27
test	922±31	942±22	941±24*	957±19*
Erythrocytes, immature
control	42±16	22±S*	30±17	26±10*
test	42±16	12±8*	13±4*	9±6*
Lymphocytes
control	44±23	43±14	74±18*	42±24
test	44±23	40±23	40±18	28±11*
Thrombocytes
control	10.7±10.3	10.8±11	30.7±18.6*	10.0±10.0
test	10.7±10.3	5.6±4.5	6.6±7.3	6.0±6.6
Neutrophils, mature
control	0.3±0.7	0.1±0.3	0.5±0.8	0.5±0.6
test	0.3±0.7	0.1±0.3	0.1±0.3	0.1±0.3
Neutrophils, immature
control	1.4±2.3	0.5±1.0	0.7±0.8	0.2±0.5
test	1.4±2.3	0	0.2±0.6	0.1±0.3

Validity criteria fulfilled:

not applicable

Conclusions:

In partial chronic (3-mo) testing with 10-13 cm rainbow trout (Oncorhynchus mykiss) in soft water at 10 °C, dissolved ozone (O3), at 2 µg/L caused no significant biological damage while 5 µg/L caused some gill pathological changes and reduced feeding behavior. Accordingly, 2 µg/L is suggested as a provisional maximum safe exposure level, pending completion of life cycle studies.

Executive summary:

Due to the thermodynamic instability of ozone and the unavailability of feedback control systems, it was not possible to achieve completely stable, graduated, ozone exposure levels over long time periods and therefore only partial chronic (3-mo) tests could be carried out at 2 ozone levels. Although this can be evaluated as a weakness, it is inherently difficult to execute chronic tests with ozone. The fish need to be fed such that they can grow while trying not to increase the ozone demand of the water too much. The study recorded many physiological parameters of health and growth, and the 2 ozone levels, although quite close to each other, showed significantly different effects. As such the study is a clear indication of the potential sub-lethal effects already appearing at very low dissolved ozone concentrations (5 µg/L). Accordingly, 2 µg/L is suggested as a provisional maximum safe exposure level, pending completion of life cycle studies. These results were obtained based on non-specific ozone measurements, meaning that other oxidative species (free chlorine or bromine) are measured too. But given that soft laboratory water was used, this is probably negligible.

Reference 2

Endpoint:

long-term toxicity to fish, other

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Juvenile turbot (Psetta maxima, L.) were exposed to Ozone produced Oxidants (OPO) concentrations of 0.06, 0.10 and 0.15 mg/l for 21 days. Gills were analysed for histopathological alterations and mRNA expression of heat shock protein 70 (hsp70), hsp90 as well as glutathione-S-transferase (GST) were determined in the gills and the liver after 1 d, 7 d and 21 d.

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

other: Psetta maxima

Details on test organisms:

TEST ORGANISM
- Common name: turbot
- Strain: Psetta maxima, L.
- Source: commercial turbot hatchery
- Age at study initiation: Juvenile
- Length at study initiation (length definition, mean, range and SD): 12.2 ± 1.0 cm TL
- Weight at study initiation (mean and range, SD): 36.4 ± 8.2 g wet weight
- Feeding during test: to apparent satiation (accounting for approximately 2% of body weight) in 48 h intervals.
- Food type: commercial pellet feed (DAN-EX 1562, 1.5 mm; Dana Feed)

- Acclimation period: 10 days

Test type:

other: recirculation system

Water media type:

saltwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

no

Test temperature:

temperature: 14.29 ± 0.45 °C

pH:

7.57 ± 0.07

Dissolved oxygen:

10.48 ± 0.16 mg/l

Salinity:

18.10 ± 0.25

Nominal and measured concentrations:

Measured OPO concentrations ±SD: 0.06 ± 0.01, 0.10 ± 0.01 and 0.15 ± 0.02 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 200 l with individual biofilter and foam fractionator
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: fiberglass, filled with 150 l filtered natural seawater
- Recirculation rate of test solution: 600 l/h via vertical spray bar and supply of ozone was controlled and regulated by a redox potential controller using the redox potential as proxy for the total oxidant concentration.
- No. of organisms per vessel: 23
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: filtered natural seawater
- Intervals of water quality measurement: Physical and chemical water parameters (temperature, salinity, pH, dissolved oxygen, total ammonia-N, nitrite-N) were measured at 2 day intervals.

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 12:12 L:D photoperiod

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Gills histology and gill and liver gene expression (hsp70, hsp90 and GST) after 1, 7 and 21 days of OPO exposure. Expression of housekeeping genes (act, ß2m and pol a) after 1 and 21 days in gills and liver.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: Ozone incl. Ozone Produced Oxidants

Basis for effect:

other: gill histology

Remarks on result:

other: mRNA expression returned to basic levels on day 21 regardless the actual OPO concentration, suggesting a collapse of adaptive mechanisms as a possible explanation for the observed tissue damage

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

other: Ozone, incl. Ozone Produced Oxidants

Basis for effect:

other: gill histology

Remarks on result:

other: mRNA expression returned to basic levels on day 21 regardless the actual OPO concentration, suggesting a collapse of adaptive mechanisms as a possible explanation for the observed tissue damage

Details on results:

Histology:
Lamellar clubbing, hypertrophy and hyperplasia occurred in all specimens analysed. Necrotic abnormalities were observed in 69.4% of the investigated specimens and were also present in controls. The total amount of affected lamellae remained almost stable in control animals, showing a slight increase in hyperplasia at day 21. In OPO-exposed turbot the amount of gill filaments showing no histopathological damage decreased steadily over the experiment with the 0.10 and the 0.15 mg/l treatment showing the most pronounced changes.

Housekeeping genes:
act, ß2m no effects; pol a significantly increased as day 1 after exposure.

Liver gene expression:
HSP70, HSP90 and GST: increased compared to control at days 1 and 7 and return to basic levels at day 21; no differences between the different OPO treatments.

Gill gene expression:
HSP70 and HSP90: dose related increase compared to control at days 1 and 7. Significantly different at 0.10 mg/l (HSP90 at day7) and 0.15 mg/l (HSP70 at days 1 and 7 and HSP90 at day 1).
GST: dose related increase compared to control at day 7. Significantly different at 0.10 mg/l.

Reported statistics and error estimates:

Data are presented as mean ± standard deviation. Data were calculated as mean for each single tank and compared between replicates and treatments. When conformed, factorial ANOVAs were conducted and significance was analysed by Tukey HSD test for equal sample sizes or unequal N HSD test, respectively. If a significant interaction between the two factors “exposure time” and “OPO concentration” was observed, the effect of each single factor was investigated by one-way ANOVA. If normality or homogeneity of variances were not confirmed, even after transformation, data were analysed with the Scheirer-Ray-Hare test. Here, subsequent analyses within levels of each factor were carried out using Kruskal-Wallis test.

Validity criteria fulfilled:

not applicable

Conclusions:

Exposure of juvenile turbot to OPO concentrations of 0.10 mg/L for 21 days induced irreversible changes in the gills. Together with evidence from an earlier study by the same authors (Reiser et al 2010), it was concluded by Reiser et al (2011) that an OPO concentration of ≤ 0.06 mg/l can be suggested as a safe level for the rearing of juvenile turbot (21 d NOEC = 0.06 mg/l).

Executive summary:

To evaluate the chronic effects of sublethal OPO concentrations, juvenile turbot (Psetta maxima, L.) were exposed to OPO concentrations of 0.06, 0.10 and 0.15 mg/l for 21 days. Gills were analysed for histopathological alterations and mRNA expression of heat shock protein 70 (hsp70), hsp90 as well as glutathione-S-transferase (GST) were determined in the gills and the liver after 1 d, 7 d and 21 d. Histopathologic findings confirmed adverse effects at 0.10–0.15 mg/l, but these (necrosis, lamellar clubbing, hypertrophy, hyperplasia) could only be observed after an extended exposure (mostly 21 d), and were considered as irreversible tissue damage. Hsp70 expression in the gills was only significantly increased at the highest OPO concentration (0.15 mg/l) on 1 d and 7 d, and returned to basic levels until day 21. Hsp90 mRNA was increased at 0.10 mg/l after 7 days of exposure, and again was comparable to the control group on day 21. In contrast, elevated GSTmRNA expression was only observed on day 7 at 0.10 mg and 0.15 mg/l. Although similar trends were observed in the liver for all markers, differences were only significant in exceptional cases due to the high individual variation observed. It has to be noted that mRNA expression returned to basic levels on day 21 regardless the actual OPO concentration.

Description of key information

The lowest NOEC value after a 3 month partial chronic testing of freshwater fish juveniles to ozone reported in the scientific literature was 2 µg/L. For marine fish only a study of 21 d is available, which exposed fish to ozone and ozone produced oxidants. The derived NOEC is 60 µg/L. Both values lie below the CLP Aquatic Hazard Assessment threshold of 0.1 mg/L, hence, ozone is classified as chronic aquatic toxicity category 1.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

2 µg/L

Marine water fish

Marine water fish

Effect concentration:

60 µg/L

Additional information

Wedemeyer et al.1979 exposed juvenile 10–13 cm rainbow trout (Oncorhynchus mykiss) to dissolved ozone at 2 µg/L and at 5 µg/L in soft water at 10 °C for 3 months. The lowest exposure level (2 µg/L) caused no significant biological damage while 5 µg/L caused histological gill damages and reduced feeding behaviour. Mortality was not observed. Accordingly, 2 µg/Lwas suggested as a provisional maximum safe exposure level. The concluded NOEC for ozone in fish is ca 2 µg/l, and hence, clearly below 0.01 mg/l. Consequently ozone, being a substance which is rapidly degraded, should be classified as Chronic aquatic toxicity category 1.

Concerning seawater fish, an exposure of juvenile turbot to ozone and ozone produced oxidants of 0.1 mg/L for 21 days induced irreversible changes in the gills. Together with evidence from an earlier study by the same authors, it was concluded by Reiser et al (2011) that an OPO concentration of ≤ 0.06 mg/l can be suggested as a safe level for the rearing of juvenile turbot (21 d NOEC = 0.06 mg/l).

Classification proposal (REGULATION (EC) No 1272/2008 and amendments): Chronic aquatic toxicity category 1.