Ozone

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

Daphnid neonates (Daphnia magna), less than 24 h old, were exposed tot ozone enriched water for 48 hours at 21 and 27 °C.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

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

Vehicle:

no

Details on test solutions:

The water was recirculated and the ozone concentration was kept at a constant level. No further details on the test solution are given.

Test organisms (species):

Daphnia magna

Details on test organisms:

Neonates, less than 24h old.

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 given.

Test temperature:

21 and 27°C

pH:

No data given.

Dissolved oxygen:

No data given.

Salinity:

No data given.

Conductivity:

No data given.

Nominal and measured concentrations:

Measured concentrations at 21 °C - control, 11 ± 3 µg/L, 21 ± 5 µg/L, 57 ± 16 µ/L, 93 ± 25 µg/L, 140 ± 19 µg/L

Measured concentration at 27 °C - control, 16 ± 5 µg/L, 23 ± 5 µg/L, 42 ± 10 µg/L, 97 ± 17 µg/L, 137 ± 21 µg/L

Details on test conditions:

The animals were not fed during the exposure.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

11 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 21°C

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

16 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 27 °C

Duration:

48 h

Dose descriptor:

LC100

Effect conc.:

21 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 21°C

Duration:

48 h

Dose descriptor:

LC100

Effect conc.:

23 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 27°C

Details on results:

It was not possible to determine an LC50 from the results as the experimental regime did not include a concentration gradient beween 11 and 21 µg/L for the 21°C group and the 16 and 23 µg/L for the 21°C group.

Results with reference substance (positive control):

not applicable

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. Therefore, the values are considered a reliable indication of acute toxicity of ozone to Daphnia magna. The NOEC was established to be 11 microgram/L

Executive summary:

The acute toxicity of dissolved ozone was determined among other species to Daphnia magna (at 21 and 27°C). The results indicate that ozone is very harmful to aquatic life. The 48-h NOEC for D. magna was 11 microgram/liter (at 21°C). At concentrations of 21 µg/L (for the 21 °C) and 23 µg/L (for the 21 °C) and above 100% mortality occurred for D. magna. It was not possible to determine an LC50 from the results as the experimental regime did not include a concentration gradient beween 11 and 21 µg/L for the 21°C group or between 16 and 23 µg/L for the 21°C group.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

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: The present study tests the efficacy of individual and combined treatments of sonication, hydrogen peroxide and ozone on the inactivation of the cyst, larval, and adult life history phases of the brine shrimp A. salina. Individual treatments consisted of exposing brine shrimp to sonication, hydrogen peroxide or ozone over 5, 10, 15 and 20 min exposure time periods. Combined treatments of sonication and hydrogen peroxide, sonication and ozone, hydrogen peroxide and ozone and, sonication with hydrogen peroxide and ozone were similarly tested over 5, 10, 15 and 20 min exposure time periods.
- Short description of test conditions: Hydrogen peroxide (USP grade) and ozone concentrations in each experimental bottle were maintained at 100 ppm, with the latter produced by a generator (OREC, Osmonics Inc. Model O3DM-110) that sparged ozone into the experimental bottle throughout the treatment period. During ozone and hydrogen peroxide treatments, the experimental or control cultures were stirred using a magnetic stirrer (70 rpm). Triplicate controls consisted of culturing D. tertiolecta and A. salina under identical culture conditions and over a similar time period to cultures used in all experimental treatments. These cultures were then transferred to 2 L polypropylene bottles for a time period identical to a given experimental exposure period but not exposed to either sonication or advanced oxidants. 2.5 L glass vessels with a 2 L culture in Artifical Sea Water; 0.4 g dry cysts/vessel, larvae from 0.4 g dry cysts/vessel and adults from 0.2 g dry cysts/vessel; 3 vessels/treatment (experimental and control)/exposure period.
- Parameters analysed / observed:mortality

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

Artemia salina

Details on test organisms:

Cysts, 24-hour old nauplier larvae and 30-day old adults. Cysts were obtained from Argent Laboratories (Argentemia Grade I Gold Label).

For larval cultures, cysts ( 0.4 g dry cysts in 2.0 l) were maintained in 2.5 l glass beakers equipped with air stones and an overhead lamp to provide a constant temperature of approximately 28 centigrade. Cysts hatched into naupliar larvae within a period of 24 h and were maintained for an additional 24 h prior to use in experiments. Hatched naupliar larvae were placed on an ad libitum diet of the dried cyanobacterium Spirulina spp. (Superpetz, premium). To prepare cultures of adults, a lower concentration of 0.2 g wt dry cysts in 2 l of Artificial Sea Water (ASW) was used to allow for lower adult densities per ml ASW. Cysts were cultured as above until hatching and subsequently fed an ad libitum diet of cyanobacteria. Developing shrimp were transferred to clean glass beakers every 4 days. All adult brine shrimp experiments were conducted using 30-day-old adult shrimp.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

20 min

Remarks on exposure duration:

5, 10, 15 and 20 min exposure time periods

Post exposure observation period:

Cysts 48h; no post exposure observation period for larvae and adults.

Hardness:

No data

Test temperature:

No data

pH:

No data

Dissolved oxygen:

No data

Salinity:

288 ppt

Conductivity:

No data

Nominal and measured concentrations:

Exposure times 5, 10, 15 and 20 min. Exposure concentration 100 ppm.

Details on test conditions:

2.5 L glass vessels with a 2 L culture in Artifical Sea Water; 0.4 g dry cysts/vessel, larvae from 0.4 g dry cysts/vessel and adults from 0.2 g dry cysts/vessel; 3 vessels/treatment (experimental and control)/exposure period. Exposure times 5, 10, 15 and 20 min. Exposure concentration 100 ppm.

Reference substance (positive control):

no

Duration:

20 min

Dose descriptor:

other: mortality %

Remarks:

larvae

Effect conc.:

69 other: %

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 100pm

Duration:

20 min

Dose descriptor:

other: mortality %

Remarks:

adults

Effect conc.:

44 other: %

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 100 ppm

Details on results:

Mortality
Cysts
5 min: 14-17%
10 - 20 min: up to 34%

Larvae
37-69% (time dependent)

Adults
23-44% (time dependent, data read from figure)

Dead and desintegrated larvae and adults were counted in experimental groups and controls. Correction was made for mortality in controls.

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

Analysis of variance (ANOVA) followed, where appropriate, by Tukey’s HSD post hoc comparison tests.

Validity criteria fulfilled:

not applicable

Conclusions:

Mortality was observed in Artemia salina cysts, larvae and adults exposed for up to 20 minutes to a concentration of 100 ppm (100 mg/L) ozone. Cysts were the least sensitive and larvae were the most. Mortality in the larvae exceeded 50 %.

Executive summary:

In the frame of evaluating different methods for ballast water treatments, the toxic effect of ozone to three discrete life history phases of the brine shrimp Artemia salina, was tested. Brine shrimp cysts, nauplii, and adults were subjected to individual and combined treatments of sonication and advanced oxidants. Combined rather than individual treatments consistently yielded the highest levels of mortality in brine shrimp (100% and 95% for larvae and adults, respectively, over a 2 min exposure). For ozone only, mortality levels in brine shrimp cysts range from 14 -17 % over 5 minutes and increased to 34% over a 20 min exposure. For larvae, mortality levels were between 27 and 69% over a 5 to 20 minute exposure period and for adults mortality levels were between 23 to 44% over the same exposure period. Overall, the results show, that ozone is toxic to aquatic invertebrates after a short exposure time and that larvae are the most sensitive life stage to ozone.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

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:

Effective total residual oxidants (TRO) in coastal water was compared with artificial sea water. Toxicity experiments were conducted following treatment with a chemical reductant to confirm that oxidant toxicity could be removed if necessary to ensure environmental safety of the ozonated seawater.

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Comparative toxicity of natural waters:
For each toxicity experiment with mysid shrimp, waters were treated with ozone until a target TRO concentration of approximately 2 mg/L as bromine 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). Total residual oxidant measurements were taken from each dilution to confirm nominal concentrations. Four 250-ml replicates of each concentration in 500-ml beakers were used for each test and maintained at 20 degrees centigrade in an environmental chamber.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

Juvenile mysid shrimp (Americamysis bahia), 8 d at initiation of test were obtained from Aquatic Biosystems (Fort Collins, CO, USA). Organisms were acclimated to test laboratory conditions for 24 h before test initiation.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

No data

Test temperature:

20°C

pH:

No data

Dissolved oxygen:

No data

Salinity:

Water Salinity
Artificial seawater 30
Cape Fear (NC, USA) 33
Shannon Point (WA, USA) 32
Vallejo (CA, USA) 13
Yaquina Bay (OR, USA) 31

Nominal and measured concentrations:

2, 1, 0.5, 0.25, 0.125 mg/L (nominal)

Details on test conditions:

Tests were completed using 500-ml plastic beakers. For each toxicity experiment with mysid shrimp, waters were treated with ozone until a target TRO concentration of approximately 2 mg/L as Br2 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). Total residual oxidant measurements were taken from each dilution to confirm nominal concentrations. Four 250-ml replicates of each concentration in 500-ml beakers were used for each test and maintained at 20°C in an environmental chamber. Ten juvenile mysid shrimp were used in each replicate and fed 0.1 ml of A. franciscana at test initiation and twice daily thereafter. The mysids were examined for mortality at 24 and 48 h, and dead organisms were removed. Conductivity and ammonium concentrations of the water were measured at test initiation, and pH, dissolved oxygen, temperature, and salinity were monitored daily.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.47 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

TRO/L as bromine

Basis for effect:

mortality

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

0.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Remarks:

TRO/L as bromine

Basis for effect:

mortality

Details on results:

Comparative toxicity of natural waters:
The LC50 values for mysids after 24 h in tests using ASW and each of the site waters were not significantly different, ranging from 0.55 to 0.62 mg TRO/L as Br2 (Table 1). After 48 h, the LC50 values also were similar, ranging from 0.34 to 0.46 mg TRO/L as Br2, although mysids in water from Shannon Point (LC50, 0.46 mg/L as Br2) were slightly less sensitive than those in water from Cape Fear (LC50, 0.34 mg/L as Br2).
The 48-h data were not available for tests using water from Vallejo because of control failure.

Table 1 (adapted)
Americamysis bahia, juvenile
LC50 (mg/L) LC50 (mg/L)
24hrs 48hrs
Artificial seawater 0.58 (0.52-0.64) 0.42 (0.37-0.48)
Cape Fear (NC, USA) 0.55 (0.49–0.61) 0.34 (0.30–0.37)
Shannon Point (WA, USA) 0.61 (0.56–0.66) 0.46 (0.39–0.53)
Vallejo (CA, USA) 0.62 NA
Yaquina Bay (OR, USA) 0.58 (0.51–0.66) 0.37 (0.33–41)

no mortality when sodium thiosulfate was added.
concentrations expressed as mg total residual oxidant (TRO) per liter as bromine.
95% confidence limits within brackets

Validity criteria fulfilled:

not applicable

Conclusions:

The 24- and 48-h LC50 values for mysids in waters tested immediately following ozone treatment were 0.70 and 0.47 mg TRO/L as bromine, respectively.

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, including 3 species of aquatic invertebrate, the mysid shrimp (Americamysis bahia), and the benthic amphipods (Leptocheirus plumulosus and Rhepoxinius abronius). The test was a static design, with 5 different test concentrations, 2, 1, 0.5, 0.25 and 0.125 mg/L. Ozone was dosed just once to the test system.

The mysid shrimp (Americamysis bahia) was the most sensitive invertebrate, and the benthic amphipods (Leptocheirus plumulosus and Rhepoxinius abronius) the least sensitive of all species tested. The fish species were more sensitive than the invertebrates tested. Mortality from ozone exposure occurred quickly, with median lethal times ranging from 1 to 3 h for the most sensitive species, although additional mortality was observed 1 to 2 d following ozone exposure. 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. An LC50 of 0.47 mg/L after 48 hours was concluded for invertebrates based on the most sensitive species tested here, the mysid shrimp (Americamysis bahia).

Description of key information

The study by Leynen (1998) provided a conservative assessment of the LC50 for invertebrates at 0.01 mg/l. This value is based on the NOEC and has been used as a conservative proxy due to data limitations.
All LC values for effects of ozone/ total residual oxidant on invertebrates were substantially lower than 1 mg/l which is the upper limit for classification of a substance for acute aquatic toxicity in category 1.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.01 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

0.32 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 aquatic invertebrates were found in the public domain or in the archives of the applicant.

Two publications on the acute toxicity of ozone to aquatic invertebrates in fresh water, and one publication in sea water were found in the public domain and evaluated.

 

For fresh water invertebrates, the value of 0.011 mg ozone /L (Leynen et al, 1998; LC0, 48h) is considered the most relevant. Although no mortality was observed at this level the, LC100 was only two-fold higher, i.e. 0.021- 0.023 mg/l, in the same

experiment after 24h. This study was considered to be the most relevant for risk assessment, since the other fresh water study (Gavand, 2007) was too basic to draw firm conclusions.

For aquatic invertebrates in seawater, the value of 0.32 mg total residual oxidant/liter (Jones et al, 2006) is the lowest LC50 at the longest exposure time (48h) and is therefore considered the most appropriate for marine risk assessement.

Concluding, an LC50 for invertebrates (fresh and marine) of ca. 0.01 mg/l seems a conservative estimation based on the results by Leynen et al (1998).

 

All values were obtained in studies not performed under GLP, but are, irrespective of freshwater or seawater, all substantially lower than 1 mg/l which is the upper limit for classification of a substance for acute aquatic toxicity in category 1.

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