Ozone

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

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

Remarks:

Peer reviewed scientific publication

Qualifier:

according to guideline

Guideline:

other: EPA standard bioassay procedure

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: no (in an effort to minimize fluctuations in ozone demand to help stabilize exposure levels).

ACCLIMATION
- Acclimation period: at least 30 d before each test.
- Acclimation conditions : laboratory water supply, the fish could not be acclimated to the test troughs before beginning the 96-h LC50 determinations, but the water flows in the holding tanks were similar to those of the 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:

96 h

Hardness:

total hardness: 20 mg/L (as CaC03)

Test temperature:

10°C

pH:

6.8

Dissolved oxygen:

11 mg/L

Nominal and measured concentrations:

2, 7, 29 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 acute toxicity tests, initial 10 fish blood and gill tissue samples were obtained and groups of 60 fish then randomly distributed to duplicate test troughs. Additional 10 fish samples were taken at 4, 24, 48, 72, and 96 h from one of the replicates during the exposure period. Moribund fish were sampled whenever possible. Death was defined as cessation of opercular movement.

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: from ca 2 microg/L - ca 29 microg/L.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

9.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 8.1 - 10.6 µg/L

Details on results:

- Behavioural abnormalities: not reported
- Observations on body length and weight: not reported
- Other biological observations: typical pathological changes in gill tissue by acute ozone exposure at 7 and 29 µg/L; severe, generalized,
stress response at 7 and 29 µg/L
- Mortality of control: <10 %
- Other adverse effects control: not reported
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

An ozone toxicity curve (Fig. 2) yielded a 96-h LC50 and 95% confidence interval estimate of 9.3 (8.1-10.6) microg/L O3, with an acute lethal threshold of about 8 microg/L.
Median lethal ozone concentrations were calculated and the resulting toxicity curve determined by the Fisheries Bioassay Laboratory, Montana State University, using a trimmed Spearman-Karber computer program (Hamilton etal.1971).

Sublethal observations / clinical signs:

To help determine the cause of death and provide a background for future forensic diagnoses, physiological damage occurring during the acute exposures was monitored. At the lowest ozone level (2µg/L) there were no noticeable changes even after 96-h exposures. At 7 µg/L, hyperplasia of the lamellar epithelium was extensive by 96 h with some separation from the supporting elements. At the higher exposure level (29 µg/L) marked epithelial degeneration had occurred by 24 h and mortality was virtually complete within about 48 h. Survivors were moribund and showed very extensive cellular damage with severe hypertrophy, hyperplasia, epithelial degeneration, and necrosis. To help clarify the degree to which the progressively severe gill pathological changes became physiologically limiting, sequential blood chemistry profiles as a function of dose and contact time were determined. Hemoglobin and hematocrit levels increased significantly (p = 0.05) and remained elevated at ozone exposures of 7 µg/L and above suggesting reduced oxygen uptake due to gill damage, and severe hemoconcentration, since frank polycythemia would be unlikely to develop in such a short time period.

Peripheral blood smears revealed generally increased numbers of immature erythrocytes, indicating an increase in hematopoiesis. Ionoregulation was evidently compromised at the higher exposure levels, as indicated by the persistant plasma electrolyte imbalances that occurred (Fig. 5A, B). A serious hyponatremia and hypochloremia developed within 4 h at 29 J1.g/L 0 3 which reached potentially lethal limits within 24-48 h. On the basis of case history data, we consider declines in plasma chloride and sodium to levels below ~ 100 meq/ L to be life-threatening for salmonids. At the 7 µg/L O3 exposure level, a significant (p = 0.05) though biologically mild hyponatremia and hypochloremia developed, which was correlated with the gill histological changes. The 2 µg/L O3 acute exposure level caused no blood electrolyte imbalances or gill histological changes. That ozone exposure also causes a severe, generalized, stress response was illustrated by the rapid, intense hyperglycemia that developed. Again, the stipled area shows the approximate normal range limits. Blood sugar levels were abnormal within 4 h at the 29 µg/L O3 levels, and within 24 h mortality was essentially complete. At 7 µg/L O3 , hyperglycemia was severe by 24 h and potentially lethal by 96 h. At the 2 µg/L ozone level, blood glucose values remained essentially within the control,range although they increased somewhat after the 1st d of exposure.

Validity criteria fulfilled:

not applicable

Conclusions:

The exposure of the animals was continuous and monitored, which is a basic requirement for this type of experiments with ozone. The LC50 values therefore are considered a reliable indication of acute toxicity of ozone to juvenile fish. The 96-h LC50 (95% confidence interval) was 9.3 (8.1 -10.6) microg/L.

Executive summary:

An acute toxicity curve for dissolved ozone (O3) in soft water at 10°C, using 10-13-cm rainbow trout (Oncorhynchus mykiss) as the test species was calculated. The 96-h LC50 (95% confidence interval) was 9.3 (8.1 -10.6) µg/L. The lethal threshold level was about 8 µg/L. Death apparently results from massive destruction of the gill lamellar epithelium together with a severe hydro mineral imbalance.

Reference 2

Endpoint:

short-term toxicity to fish

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:

Larvae of three fish species were exposed to ozone enriched water for 48 hours. Two replicates of 10 larvae were exposed to each of five test concentrations of ozone.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

At regular time intervals. Interval length and number and sampling method not specified.

Vehicle:

no

Test organisms (species):

other: Cyprinus carpio, Leuciscus idus, and Clarias gariepinus

Details on test organisms:

Fish larvae were collected from a hatchery (Cyprinus carpio, Leuciscus idus) or obtained from the laboratory culture (Clarias gariepinus).

Mean total length (cm) and total wet weight (mg)
Cyprinus carpio: 1.1 ± 0.1 cm, 17 ± 3 mg
Leuciscus idus: 1.7 ± 0.2 cm, 50± 8 mg
Clarias gariepinus: 1.1 ± 0.1 cm, 11 ± 2 mg

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

No data.

Test temperature:

Cyprinus carpio: 27degrees centigrade.
Leuciscus idus: 27degrees centigrade.
Clarias gariepinus: 32 degrees centigrade.

pH:

No data given.

Dissolved oxygen:

No data given.

Salinity:

No data given.

Conductivity:

No data given.

Nominal and measured concentrations:

No data given.

Details on test conditions:

The animals were not fed during the exposure.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

35 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: mean of different LC50

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

31 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Cyprinus carpio

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

39 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Leuciscus ide

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

35 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Clarias gariapinus

Details on results:

Species and LC50 in µg/L (duplicate experiments in two species). 95 % confidence interval between brackets.

Binomial method
Cyprinus carpio: 31 (26-36) and 44 (41-61)
Leuciscus idus: 36 (27-48) and 39 (33-46)
Clarias gariepinus: 35 ( 21-64)

Probit analysis.
Cyprinus carpio: 31 (28-33) and 44 (43-45)
Leuciscus idus: could not be determined.
Clarias gariepinus: 37 ( 29-46)

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

LC50 values with 95 % confidence limits were calculated with the binomial method and probit analysis.

Validity criteria fulfilled:

not applicable

Remarks:

no claim that study was performed according to a guideline

Conclusions:

The exposure of the animals was continuous and monitored, which is a basic requirement for this type of experiments with ozone. The LC50 values therefore are considered a reliable indication of acute toxicity of ozone to fish larvae.

Executive summary:

The acute toxicity of dissolved ozone was determined for fish larvae of three species [Cyprinus carpio (at 27°C), Leuciscus idus (at 27°C) and Clarias gariepinus (at 32°C)] and to Daphnia magna (at 21 and 27°C). The results indicate that ozone is very harmful to aquatic life. The LC50 values for the larvae are as follows:

LC50 (48h, C. carpio) = 31 (95% CL: 26-36) µg/L

LC 50 (48 h, L. idus) = 39 (95% CL: 33-46) µg/L

LC50 (48 h, C. gariepinus) = 35 (95% CL: 21-64) µg/L

The mean 48-h LC50 value for the fish larvae amounts to about 35 µg/L, while the 48-h NOEC for D. magna was 11 µg/L (at 21°C).

Reference 3

Endpoint:

short-term toxicity to fish

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:

- Principle of test: Effective total residual oxidants (TRO) in ozonated seawater was compared with ozonated artificial sea water (ASW). Comparative toxicity tests with topsmelt and sheepshead minnows, using ASW and water from Yaquina Bay.
- Short description of test conditions: Batch ozone toxicity experiments were conducted to simulate the ozone exposure used onboard the S/T Tonsina. Tests with larval A. affinis, juvenile C. variegatus, and adult R. abronius were conducted in 20-L glass aquaria, whereas experiments with adult mysid shrimp and adult L. plumulosus were conducted in 10-L glass aquaria. Small pieces of nylon mesh were placed as substrate in aquaria used to conduct toxicity tests with L. plumulosus and R. abronius. All experiments used a total of five chambers, each containing 10 organisms, with one chamber tested per treatment. Chambers were maintained at 23 ± 2°C for all organisms except R. abronius, which were maintained at 15 ± 2°C. Total gas flow rates for 20-L chambers were 97.5, 63.2, 38.6, and 20.0 ml/min. These flow rates corresponded to nominal ozone supply rates of 0.43, 0.28, 0.17, and 0.09 mg O3/ L/min, respectively. Controls received compressed, ambient air at 97.5 ml/min (i.e., maximum flow rate). Total gas flow rates for 10-L chambers were 38.6, 28.3, 20.0, and 13.1 ml/ min (0.34, 0.25, 0.17, and 0.11 mg O3/L/min, respectively; control air flow rate, 38.6 ml/min). Experiments were run for a maximum of 5 h to simulate ozone exposure durations used during S/T Tonsina field trials. Total residual oxidant measurements were recorded with biological observations (mortality and motility of survivors) at 0.5, 1, 2, 3, 4, and 5 h following test initiation. Experiments were terminated within the 5-h exposure period if all organisms in a treatment died.
- Parameters analysed / observed: mortality and motility of survivors

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: waters were treated with ozone until a target TRO concentration of approximately 1 mg/L as Br2 (topsmelt) and 0.25 mg/L as Br2 (sheepshead) was reached. Serial dilutions with nonozonated water were achieved at concentrations of 100 (ozonated water only), 50, 25, 12.5, 6.25, and 0% (nonozonated water only). Topsmelt. 100% = 1 mg/L as Br2; sheepshead, Yaquina Bay sample: 100% = 0.25 mg/L as Br2. Both topsmelt and sheepshead minnows were tested in 400-ml replicates and fed 0.2 ml of A. franciscana twice daily. Because of specifications provided in U.S. Environmental Protection Agency protocol 600/4-90/027F [31], experiments with sheepshead minnows were conducted for 96 h instead of 48 h. After 48 h, test solutions were renewed with freshly prepared exposure solutions; median lethal concentrations (LC50) were calculated at 24-h intervals from 0 to 96 h.
- Sampling method: Total residual oxidant measurements were taken from each dilution to confirm nominal concentrations.

Vehicle:

no

Details on test solutions:

Because previous experiments suggested that both organisms were more sensitive to ozonated seawater, lower maximum TRO concentrations were used than for invertebrate tests: topsmelt, 100% = ca 1 mg/L as Br2; sheepshead, Yaquina Bay sample, 100% = ca 0.25 mg/L as Br2.

Test organisms (species):

other: Atherinops affinis, Cyprinodon variegatus

Details on test organisms:

Atherinops affinis (larvae, 11-12 d) and Cyprinodon variegatus (juvenile, 3-4d), obtained from Aquatic Biosystems (Fort Collins, CO, USA). Organisms were acclimated to test laboratory conditions for 24 h before test initiation. Animals were fed Artemia franciscana twice daily.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

A. affinis: 48 hrs; C. variegatus 96 hrs.

Post exposure observation period:

no

Hardness:

No data

Test temperature:

20°C

pH:

No data

Dissolved oxygen:

No data

Salinity:

28-31 practical salinity units (PSU)

Conductivity:

No data

Nominal and measured concentrations:

Serial dilutions with nonozonated water were achieved at concentrations of 100 (ozonated water only), 50, 25, 12.5, 6.25, and 0% (nonozonated water only). Topsmelt. 100% = 1 mg/L as Br2; sheepshead, Yaquina Bay sample: 100% = 0.25 mg/L as Br2.

Details on test conditions:

Tests with larval A. affinis and juvenile C. variegatus were conducted in 400 mL water in 500 ml plastic beakers. Six concentrations, four replicates for each concentration. Tests were performed in artificial sea water and natural sea water. A. affinis was exposed for 48 hrs. and C. variegatus was exposed for 96 hrs. After 48 hrs test solutions were renewed with freshly prepared solutions. Test was repeated but with addition of 0.005 M sodium thiosulfate to the solution before the organisms were added.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Remarks:

C. variegatus

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Yaquina Bay (OR, USA) seawater

Duration:

48 h

Dose descriptor:

LC50

Remarks:

A. affinis

Effect conc.:

0.26 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Yaquina Bay (OR, USA) seawater

Duration:

96 h

Dose descriptor:

LC50

Remarks:

C. variegatus

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Artificial seawater

Duration:

48 h

Dose descriptor:

LC50

Remarks:

A. affinis

Effect conc.:

0.34 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: artificial seawater

Details on results:

- Behavioural abnormalities: not reported
- Observations on body length and weight: not reported
- Other biological observations: not reported
- Mortality of control: none

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

Trimmed Spearman-Karber method or by linear interpolation if acceptable trim values were exceeded.

Sublethal observations / clinical signs:

A. affinis (topsmelt):

Toxicity tests in artifical seawater (ASW) and natural seawater from Yaquina Bay showed that topsmelt were less sensitive than sheepshead minnows over 24 and 48 h in both cases (24-h LC50, 0.34 mg/L as Br2; 48-h LC50, 0.26 mg/L as Br2) (Table 1).

C. variegatus (sheepshead minnow):

Toxicity tests with sheepshead minnows in artifical seawater (ASW) resulted in 100% mortality at TRO concentrations equal to or less than 0.23 mg/L as Br2 after 24 h (LC50, 0.17 mg/L as Br2) (Table 1). However, tests conducted using natural seawater from Yaquina Bay (100% = ca 0.23 mg/L as Br2) yielded a LC50 value comparable to that observed in ASW (0.18 mg/L as Br2 at 24–96 h).

     

Table 1: Median lethal concentrations (LC50) for juvenile topsmelt (Atherinops affinis) and sheaphead minnow (Cyprinodon variegatus) after 24, 48, 72, and 96 h of exposure to ozonated seawater^a;

^a Values in parentheses represent the 95% confidence interval. NA = not applicable; TRO = total residual oxidant. ^b No 95% confidence interval available.

		LC50 (mg TRO/L as Br2)
Species	Seawater	24 h	48 h	72 h	96 h	With addition of 0.005 M Na2S2O3
Atherinops affinis	Artificial seawater	0.34 (0.32–0.36)	0.34 (0.32–0.36)	NA	NA	No mortality
Atherinops affinis	Yaquina Bay (OR, USA)	0.26 (0.25–0.27)	0.26 (0.25–0.27)	NA	NA	No mortality
Cyprinodon variegatus	Artificial seawater	0.17^b	0.17^b	0.17^b	0.17^b	No mortality
Cyprinodon variegatus	Yaquina Bay (OR, USA)	0.18 (0.17–0.19)	0.18 (0.17–0.19)	0.18 (0.17–0.19)	0.18 (0.17–0.19)	No mortality

           

All toxicity tests conducted with topsmelt and sheepshead minnows after the addition of Na2S2O3 resulted in no mortality at any tested TRO concentration.

These data confirm that ozonated ASW is toxic to marine organisms, but ozone per se is not likely to be the chemical species directly responsible for toxicity. Ozone has a very short chemical half-life in seawater (ca. 5 s) [43], and it disappears quickly following the introduction of ozone gas. The presence of high concentrations of bromide ion (Br-) changes the chemistry of ozone decomposition when compared to freshwater systems. Ozonation of seawater oxidizes bromide ion to bromine (hypobromous acid [HOBr] and hypobromite ion [OBr-] with a pKa of 8.8 as HOBr/OBr-) that may lead to bromate ion (BrO3-). Additionally, the presence of organic material may result in reactions with HOBr to produce bromoform (CHBr3).

Validity criteria fulfilled:

not applicable

Conclusions:

The vertebrates Atherinops affinis and Cyprinodon variegatus were the more sensitive than the invertebrate Americamysis bahia which was tested in parallel under the same conditions. The LC50 values in the vertebrates ranged from 0.17 to 0.34 mg TRO/L (total residual oxidant) as bromine. Cyprinodon variegatus was slightly more sensitive. No differences in LC50 in ozonated artificial sea water and natural sea water was observed in this species. Ozonated natural sea water was slightly more toxic in Atherinops affinis. Addition of sodium thiosulfate removed all total residual oxidant.

Executive summary:

The use of ozone as an oxidant to eliminate nonindigenous species from ballast while ships are in transit has been considered. The toxicity of ozone in artificial seawater (ASW) was determined for five species of marine organisms in short-term (5 h) batch exposures. Larval topsmelt (Atherinops affinis) and juvenile sheepshead minnows (Cyprinodon variegatus) were the most sensitive to oxidant exposure, and the mysid shrimp (Americamysis bahia) was the most sensitive invertebrate.

Because ozone does not persist in seawater, toxicity likely resulted from bromide ion oxidation to bromine species (HOBr and OBr), which persist as residual toxicants after at least 2 d of storage. Total residual oxidant (TRO; as Br2) formation resulting from ozone treatment was measured in ASW and four sitespecific natural seawaters. The rate of TRO formation correlated with salinity, but dissolved organic carbon and total dissolved nitrogen did not affect TRO concentrations. Acute toxicity tests with each water over 48 h using mysid shrimp, topsmelt, and sheepshead minnows yielded results similar to those of batch exposure.

Reference 4

Endpoint:

short-term toxicity to fish

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:

according to guideline

Guideline:

other: American Public Health Association, 1971. Standard methods for the examination of water and wastewater. 13th ed. Amer. Publ. Health Assoc., Washington.

Principles of method if other than guideline:

Lepomis macrochirus were exposed to ozonated water in a continuous flow system for 24 hours after which they were observed for mortality during a 7 day period.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

A 50 cm cuvette was used to measure concentrations less than 0.03 ppm; a cell with a 20 mm light path was employed to evaluate concentrations exceeding 0.03 ppm. Ozone concentrations were determined every 2 h; the mean of the 12 resulting measurements were considered the tested concentration. A 24-h control run was conducted in the same facilities under identical conditions of water flow.

Vehicle:

no

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

Lepomis macrochirus (mean weight 3.82g) were exposed to ozone after several weeks of acclimatisation. During acclimatisation fish were fed chopped fish and commercial trout chow.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

24 h

Hardness:

62 ppm as Calcium carbonate

Test temperature:

9.0 - 13.0 degrees centigrade

pH:

7.1 - 7.2

Dissolved oxygen:

16 - 18 ppm

Nominal and measured concentrations:

Measured: 0.00, 0.01, 0.02, 0.05, 0.06, 0.07 and 0.09 ppm

Details on test conditions:

Tygon tubing was used to transport ozone from the generator to a mixing column--a vertically situated 183-cm section of 15 cm PVC pipe acting as a counter-current exchanger. A continuous flow of charcoal-filtered city water was ozonated and conveyed to the experimental raceway. A second mixing column, similarly designed, was used to mix water with either pure oxygen or compressed air. Water from this mixing column was conveyed to a second raceway where the experiments were conducted. The wooden raceways, 236 cm long and 30 cm wide, were covered by two coats of epoxy paint and filled to a depth of 12 cm. Water from the mixing columns entered at the head of each raceway at approximately 1.9 litres/min and flowed out from a standpipe at the opposite end.

Ten fish per concentration were confined within 30 by 45 cm plastic screen baskets. By confining the fish within restricted areas two bioassays could be conducted simultaneously at different concentrations in the same raceway.

Reference substance (positive control):

no

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 0.05 -0.06 mg/L

Details on results:

Survival (%) Ozone (ppm) S.D. (ppm) Range (ppm)
100 0.00 0.00 0.00 - 0.00
100 0.01 0.01 0.02 - 0.01
100 0.02 0.01 0.04 - 0.01
90 0.05 0.01 0.07 - 0.04
40 0.06 0.01 0.08 - 0.04
0 0.07 0.02 0.12 - 0.05
10 0.09 0.02 0.12 - 0.06

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

The concentration resulting in survival of 50% of the test animals was determined by probit analysis (Finney, 1971; Bliss, 1952)

Conclusions:

The LC50, 24h of ozone in Lepomis macrochirus is 0.06 +/- 0.00 ppm.

Executive summary:

Ozone toxicity to bluegill (Lepomis macrochirus) was determined using standard bioassay procedures. In initial work bluegill were exposed to water containing ozone for 24 h; the LC50 was calculated at 0.06 ppm, equivalent to 60 µg/L.

Additional experiments were conducted. A second experiment simulated "shock defouling" -- the periodic dosing of power plant cooling water with high biocide concentrations to prevent biofouling. The LC50 for a series of six 30-min exposure periods spaced 8 h apart was 0.32 ppm. A third experiment attempting to determine the effects of sublethal concentrations resulted in the death of 9 of 15 bluegill after 4 weeks of exposure to 0.01 ppm of ozone. No controls died in any experiment. It was concluded that ozone is highly toxic to bluegill. Safe limits for long-term exposure probably lie well below 0.01 ppm.

Reference 5

Endpoint:

short-term toxicity to fish

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:

Lepomis macrochirus were exposed to ozonated water in a continuous flow system for up to 10 hours. Blood samples were taken immediately following exposure, whereafter blood samples were taken and the osmolality of the blood serum was determined.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

no data

Vehicle:

no

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

Lepomis macrochirus ssp. rafinesque, acclimated before test, healthy during acclimation.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

6 h

Post exposure observation period:

n/a

Hardness:

no data

Test temperature:

no data

pH:

no data

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

0.09 ppm

Details on test conditions:

Tygon tubing was used to transport ozone from the generator to a mixing column--a vertically situated 183-cm section of 15 cm PVC pipe acting as a counter-current exchanger. A continuous flow of charcoal-filtered city water was ozonated and conveyed to the experimental raceway. A second mixing column, similarly designed, was used to mix water with either pure oxygen or compressed air. Water from this mixing column was conveyed to a second raceway where control experiments were conducted. The wooden raceways, 236 cm long and 30 cm wide, were covered by two coats of epoxy paint and filled to a depth of 12 cm. Water from the mixing columns entered at the head of each raceway at approximately 1.9 litres/min and flowed out from a standpipe at the opposite end into a sewer.

Immediately after exposure gill morphology was examined microscopically.

5 animals / exposure

Reference substance (positive control):

no

Details on results:

Exposure to ozone (0.09 ppm) caused the destruction and loss of epithelial cells covering the gill filaments. Damage was severe and led to exposure and destruction of lamellae and frequent loss of the entire tip of the filament. These effects were primarily confined to the distal portion of the
filament, leaving the base relatively unscathed. Gills of experimental fish also appeared to be bleached and lacking in blood. None of these effects was observed in control fish.

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

n/a

Validity criteria fulfilled:

not applicable

Conclusions:

Exposure to 0.09 ppm ozone rapidly results in damage to the gills which might be the underlying cause of the decrease in osmolality which was observed by the same investigators.

Executive summary:

Ozone causes gross damage to the gills. Extensive fungal infections often followed exposure to ozone, suggesting epithelial damage. An abrupt decrease in blood serum osmolality was detected in fish exposed to ozone. Mortality occurred when osmolality dropped approximately 50 mOsm below initial values

Description of key information

Lowest LC50 value after a 96 h continuous exposure of fish juveniles to ozone reported in the scientific literature was ca 0.008 mg/L.

Ozone has to be classified as acute aquatic toxicity category 1.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.008 mg/L

Marine water fish

Marine water fish

Effect concentration:

0.017 mg/L

Additional information

No studies which were performed according to internationally accepted guidelines (OECD, EC, EPA etc…) for evaluation of the acute toxicity of ozone to fish were found in the public domain or in the archives of the applicant.

Four appropriate publications on the acute toxicity of ozone in freshwater fish, and one publication on marine fish were found in the public domain. All studies are peer reviewed, well documented and meet generally accepted scientific principles.

The lowest LC50 value reported in the review was an LC50 96-h of 0.0093 mg/l in rainbow trout (Oncorhynchus mykiss), by Wedemeyer et al (1979). The reported LC50 of 0.0093 mg/l (0.0081 – 0.0106 mg/l 95% confidence interval) was also the lowest of the five original papers, in combination with the highest exposure time (96h). Since the study was evaluated to be of adequate validity, this study was appointed as key study for risk assessment. It can be concluded that the lowest lethal level LC50 (96h) is ca 0.008 mg/l.

Based on the study by Jones et al (2006) on marine fish (seawater), the value of 0.17 mg total residual oxidant/liter is considered the most appropriate for marine fish risk assessment. This is the lowest LC50 at the longest exposure time (96h) reported by the authors.

All LC50 values after a 24 to 96 hour continuous exposure of fish larvae or juveniles to ozone were below 1mg/L. The upper limit for classification of a substance for acute aquatic toxicity in category 1 is 1 mg/L. Therefore, we propose to classify ozone as acute aquatic toxicity category 1.

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