Ozone

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-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:

Chronic exposure experiments where juvenile Litopenaeus vannamei are exposed to sublethal Ozone Produced Oxidants (OPO) concentrations (±SD) of 0.06 (±0.010), 0.10 (±0.013) and 0.15 (±0.015) mg/l for 21 days.

GLP compliance:

not specified

Analytical monitoring:

yes

Vehicle:

no

Test organisms (species):

other aquatic crustacea: Litopanneus vannamei

Details on test organisms:

Juvenile Pacific white shrimp (L. vannamei) were obtained from Ecomares GmbH (Germany) and stocked to 12 identical recirculation systems 7 days prior to experiments.
Feeding: Shrimp were fed ad libitum in 48 h intervals to minimize fluctuations in ozone demand and therefore stabilize exposure levels.
Monitoring: Shrimp were checked for behavioural alterations such as loss of equilibrium and lethargy by observing behaviour in regular time intervals. Feeding activity of shrimp was assessed by monitoring the shrimp's response during and within 20 min after feeding. Dead animals were removed regularly, but could not be analysed for physiological and histological alterations due to rapid decomposition caused by cannibalism.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

No. Surviving shrimp were killed in ice water and checked for morphological abnormality.

Test temperature:

27.3 (±0.27) °C

pH:

7.4 (±0.21)

Dissolved oxygen:

8.4 (±0.16) mg/l

Salinity:

18.5 (±0.30) ppt

Nominal and measured concentrations:

OPO concentrations (±SD) of 0.06 (±0.010), 0.10 (±0.013) and 0.15 (±0.015) mg/l

Details on test conditions:

- Chronic toxicity tests were performed in 12 identical experimental recirculation systems, located in a temperature controlled lab to ensure constant environmental conditions. Each experimental system consisted of a 200 l fiberglass tank with biofiltration and foam fractionation operated in by-pass. Tanks were filled with approximately 150 l of filtered natural seawater and covered with transparent acrylic glass to avoid animal losses.
- The ozone-enriched air was injected into the seawater through a porous lime stone diffuser at the bottom of the foam fractionator (Model 1 AH 1100, Erwin Sander ElektroapparatebauGmbH, Germany), which served as a contact chamber between water and gaseous ozone.
- Recirculating water entered the foam fractionator at the top and flowed downward–past the uprising bubbles–creating a counter current exchanger which maximized diffusion of ozone into the water. The retention time in the foam fractionator was set to approximately 1 min.
- Ozonated water was discharged into the shrimp-tanks with high flow rates (600 l/h) and dispersed by perforated pipes over the whole water column. The induced circular current caused a complete mixing of inflow-water and therefore enabled identical OPO concentrations over the whole water body.
- For the chronic exposure experiment OPO concentrations sublethal to juvenile L. vannamei were selected based upon results of the acute toxicity study (data not shown).
- Two replicates of 15 shrimp each were exposed to each of the three OPO concentrations (±SD) of 0.06 (±0.010), 0.10 (±0.013) and 0.15 (±0.015)mg/l for 21 days.
- Control: 30 individuals, divided in two replicate groups, were maintained under identical conditions but without ozonation for the same period of time.

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: OPO

Basis for effect:

mortality

Remarks on result:

other: ± 0.010

Details on results:

Throughout the long-term toxicity test all shrimp in control and 0.06 mg/l tanks survived and did not show any obvious behavioural abnormalities. Even at higher OPO concentrations no behavioural impairment such as loss of equilibrium, lethargy or reduced feeding activity could be observed. However, an obvious increase of cannibalistic behaviour in shrimp exposed to the 0.10 and 0.15mg/l treatments was evident and mortality levels reached 47% and 43% after 21 days of exposure, respectively.
However, mortality did not appear until day 12 and 9 in 0.10 and 0.15 mg/l treatments, respectively. The mortality response surface is shown in Fig. 1.
After the 21 day exposure 69% and 35% of the survivors showed clear indications of soft shell syndrome at OPO concentrations of 0.10 and 0.15 mg/l, respectively. The affected shrimp had a soft, paper-like carapace with a gap between muscle tissue and exoskeleton (see Fig. 2).

Results with reference substance (positive control):

-

Validity criteria fulfilled:

not applicable

Conclusions:

Long-term exposures of juvenile Pacific white shrimp (L. vannamei) to "ozone produced oxidants" (OPOs) at 0.10 and 0.15 mg/l induced incidence of soft shell syndrome which led to mortalities due to cannibalism. Juvenile L. vannamei tested against 0.06 mg/l of ozone-produced oxidants survived the 21 day exposure and did not show any observable impairment (21 d NOEC = 0.06 mg/l).

Executive summary:

In marine recirculating aquaculture systems ozone, as a strong oxidant, is often used to improve water quality by reducing the pathogen load and removing inorganic and organic wastes. However, mainly when disinfection of recirculating water is desired, high ozone dosage is required, which may lead to toxicity problems for the cultured species. Acute toxicity of ozone-produced oxidants (OPO) to juvenile Pacific white shrimp, Litopenaeus vannamei, was assessed (data not shown here) and a safe level for residual oxidant concentration was calculated and further verified by chronic exposure experiments. While long-term exposure of juvenile white shrimp to an OPO concentration of 0.06 mg/l revealed no observable effect, long-term exposures to 0.10 and 0.15 mg/l induced incidence of soft shell syndrome which led to mortalities due to cannibalism. Thus, an OPO concentration of 0.06 mg/l is suggested to be the maximum safe exposure level for rearing juvenile L. vannamei.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Data waiving:

exposure considerations

Justification for data waiving:

other:

Justification for type of information:

Ozone is a gas and will move to the atmosphere after being released in the environment or will react immediately with components such as organic material and metal ions in water. Regarding the half -life in water (500 to 5000 s, see Gardoni et al., 2012 in section 5.6), long term exposure of aquatic invertebrate organisms is considered unlikely: In view of the short half-life, ozone generated for the purpose of water ozonisation can safely be expected to completely decompose still within the technical environment (i.e. in the sewerage), before reaching the STP or any water bodies. Therefore, the concentration of ozone in surface waters resulting from technical ozone generation can be considered to be negligible. Performance of long-term toxicity studies on invertebrates is not considered to be required.

Description of key information

Regarding the half-life in water (500 to 5000 s, see Gardoni et al. (2012)), long term exposure of aquatic invertebrate organisms is considered unlikely. Based on the toxicity in fish (see section 6.1), ozone is already classified in category 1 for chronic aquatic toxicity, which is the most stringent classification for this end-point. Long term toxicity testing on aquatic invertebrates would not be of added value. Though, a published study (Schroeder et al., 2010) reported that long-term exposures of juvenile Pacific white shrimp (Litopanneus vannamei) to Ozone Produced Oxidants (OPOs) at 0.10 and 0.15 mg/l induced incidence of soft shell syndrome which led to mortalities due to cannibalism. Juvenile L. vannamei tested against 0.06 mg/l of ozone-produced oxidants survived the 21 day exposure and did not show any observable impairment (21 d NOEC = 0.06 mg/l). These data support the classification as category one for chronic aquatic toxicity.

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