Ozone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Qualifier:

no guideline followed

Principles of method if other than guideline:

The unicellular green alga Dunaliella tertiolecta was used as model organism to study the efficacy of ozone as potential ballast water treatment . Individual ozone treatment consisted of exposing algal cells to ozone over 5, 10, 15 and 20 min exposure time periods, after which algal cell viability was determined first counting the number of remaining cells using a hemocytometer followed by vital staining techniques to evaluate the cell viability of the algal cells.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Dunaliella tertiolecta

Details on test organisms:

D. tertiolecta (Strain # CCMP1320), was cultured to stationary phase in gently aerated artificial seawater (ASW) enriched with Guillard’s f/2 mediumin 2000 ml glass flasks.

Test type:

static

Remarks on exposure duration:

5, 10, 15 and 20 min exposure time periods.

Post exposure observation period:

No

Hardness:

no data

Test temperature:

no data

pH:

no data

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

ozone concentrations in each experimental bottle were maintained at 100 ppm

Details on test conditions:

2 L polypropylene vessels; 3 vessels/treatment (experimental and control)/exposure period.
Exposure times 5, 10, 15 and 20 min. Exposure concentration 100 ppm.
During ozone treatment, the experimental or control cultures were stirred using a magnetic stirrer (70 rpm).
Triplicate controls consisted of culturing D. tertiolecta under identical culture conditions and over a similar time period to cultures used in experimental treatment. 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 ozone.

Reference substance (positive control):

no

Duration:

5 min

Dose descriptor:

LOEC

Effect conc.:

100 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality

Details on results:

Mortality
5 - 10 min: 4%
10 - 20 min: up to 40%

All individual factor experimental treatments caused significantly higher (P < 0.05) levels of mortality in algae than control treatments at all exposure time periods tested. The results are also preseented in figure 1, see below.

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

Statistical differences in mean percent mortality levels of algal cells in experimental and control treatments for all exposure time periods were determined using an analysis of variance (ANOVA) followed, where appropriate, by Tukey’s HSD post hoc comparison tests.

Validity criteria fulfilled:

not applicable

Remarks:

study was not performed according to a guideline.

Conclusions:

LOEC (5 min) ≤ 100 ppm

Executive summary:

The unicellular green alga Dunaliella tertiolecta were among other experiments used to study the efficacy of sonication as well as the advanced oxidants, hydrogen peroxide and ozone, as potential ballast water treatments. Combined rather than individual treatments consistently yielded the highest levels of mortality in algal cells (100% over a 2 min exposure) and in brine shrimp (100% and 95% for larvae and adults, respectively, over a 2 min exposure). Single ozone exposure to the alga Dunaliella tertiolecta lead to a mortality of 4% after 5 minutes, which differed significantly from the control. Therefore, it can be concluded that a LOEC is probably below or equals 100 ppm.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Qualifier:

no guideline followed

Principles of method if other than guideline:

Cultures of 3 marine algae species were treated with ozone for a short period. Subsequently the growth of the algae was measured during a 5 day period whereafter the algae were reinoculated in untreated water. Hereafter the growth was again measured during a period of 4 or 6 day in two of the three species.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

The sample size varied from 25 to 40 mL, depending upon the exposure time and consequently the total residual oxidants (TRO) levels.

Vehicle:

no

Details on test solutions:

Prior to initiating the experiments, ozone flow rates were evaluated in test flasks, which contained only filtered seawater, and the rate was adjusted to provide a maximum TRO value of approximately 0.90 mg/L.

Test organisms (species):

other: see details on test organisms

Details on test organisms:

Nannochloropsis oculata, Isochrysis galbana and Chaetoceros gracilis. All organisms were grown in 50 L cilinder of filtered and autoclaved seawater to produce a concentration of 0.79 x 10E6 cells/mL (Nannochloropsis oculata), 0. 32 x 10E6 cells/mL (Isochrysis galbana) or 0. 31 x 10E6 cells/mL (Chaetoceros gracilis).

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Post exposure observation period:

The cultures were grown for 5 d after treatment.

Hardness:

no data

Test temperature:

25 °C

pH:

no data

Dissolved oxygen:

no data

Salinity:

no data

Nominal and measured concentrations:

N. oculata
treatment measured TRO
duration (min) (mg/L)
0 0
0.5 0.006
0.75 0.006
1.0 0.014
1.5 0.202
2.0 0.481

I. galbana
treatment measured TRO
duration (min) (mg/L)
0 0
0.5 0
1.0 0.03
1.5 0.08
2.0 0.21
3.0 0.48

C. gracilis.
treatment measured TRO
duration (min) (mg/L)
0 0
0.5 0.01
1.0 0.03
1.5 0.05
2.0 0.10
3.0 0.25

Details on test conditions:

- Six treatments, each having a different period of ozone exposure, were utilized for each experiment.
- Four replicates per treatment were used.
- Algal populations were exposed to ozone at the beginning of each experiment. Ozone exposure was carried out in 2-L Pyrex® flasks, which contained a known concentration of algae in approximately 1.8 L of pre-filtered and autoclaved seawater. A small portion of ozone was diverted via norprene tubing from a working ozone generator to the flasks. An adjustable acrylic air flow meter was used to regulate ozone flow, and a fine pore air stone (2.5 X 1.5 cm) was used to diffuse the ozone into the flasks
- Twenty-four 2-L flasks were filled from the stock culture, each with approximately 1.8 L of algae. After treatment the cultures were grown for 5 d with a 24-h photoperiod using flourescent light banks designed to provide approximately 4000 lux. Light aeration was provided for each flask and the temperature was maintained at 25 degrees centigrade.
- A 5 mL sample was taken daily fromeach flask to determine algal concentrations. Cell counts were determined by measuring the absorbance of each sample at a wavelength of 440 nm (N. oculata) or 750 nm (I. galbana and C. gracilis).
- Initial cell densities were 0. 79 x 10E6 cells/mL (Nannochloropsis oculata), 0. 32 x 10E6 cells/mL (Isochrysis galbana) or 0. 31 x 10E6 cells/mL (Chaetoceros gracilis). Four flask were used for each treatment period.

Reference substance (positive control):

no

Duration:

0.5 min

Dose descriptor:

LOEC

Effect conc.:

<= 0.06 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: TRO

Basis for effect:

cell number

Remarks on result:

other: measured one day after exposure

Details on results:

The effects of ozone treatment on three species of phytoplankton were determined by measuring the cell counts of the algae each day for 4-5 d following the day of exposure.
Cell counts for all treatments of N. oculata were significantly lower than that of the control for the first 2 d (Table 1). On d-3, the 0.5 min treatment, which consisted of cultures treated with 0.05 mg/L TRO, was not significantly different from the control. By d-4, those treated at 0.12 mg/L TRO, were not significantly lower in cell count than the control. Additionally, algae treated with 0.24 mg/L TRO had similar cell counts as the control on d-5.
Growth of ozone treated I. galbana was measured daily for 4 d (Table 2). On d-1, only the treatment which produced a TRO level of 0.05 mg/L was not significantly different from the control. Cultures that had TRO levels of 0.23 mg/L showed no significant difference in cell count on d-2, and by d-4, cultures treated with TRO at 0.34 mg/L were similar in cell count to the control, as well.
The growth response of C. gracilis is presented in Table 3. Cell counts for all the treatments were significantly lower than the control through d-2. On d-3, only the 0.5 min treatment (TRO = 0.06 mg/L) was not significantly lower. On d-4, the treatment at an initial TRO of 0.21 mg/L also was not significantly different from the control, and by d-5, the 1.5 min treatment (TRO = 0.31 mg/L) had cell counts similar to the control.

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

Two-way analysis of variance was used to determine significant differences (P <0.05) in algal growth. Daily concentrations of algae exposed to varying levels of ozone were compared to the control using Dunnett's t-test.

Table 1: Cell counts of Nannochloropsis oculata (x10⁶cells/mL) exposed to varying levels of ozone. Values represent mean (of four replicates. Values within a column having a * are significantly different from the control, using the Dunnett's t-test (P < 0.05).

Missing values (-) were below detectable limits.

Nannochloropsis oculata
	Cell counts (x 10^6/ml)
Day after treatment	d0	d1	d2	d3	d4	d5
TRO in mg/ L (treatment duration in minutes)
Control	0.79	2.00	3.86	9.35	15.40	19.20
0.05 (0.5)	0.79	1.81*	3.42*	8.72	14.46	18.13
0.12 (0.75)	0.79	1.66*	3.11*	7.66*	13.61	18.21
0.24 (1)	0.79	1.42*	2.50*	6.22*	12.09*	15.98
0.67 (1.5)	0.79	1.11*	1.60*	4.00*	8.14*	13.11*
0.92 (2)	0.79	0.74*	0.47*	1.16*	1.74*	4.44*
Pooled Standard Error		0.037	0.094	0.026	0.077	0.013

 

 

Table 2: Cell counts of Isochrysis galbana (x10⁶cells/mL) exposed to varying levels of ozone. Values represent mean (of four replicates. Values within a column having a * are significantly different from the control, using the Dunnett's t-test (P < 0.05).

Missing values (-) were below detectable limits.

Isochrysis galbana
	Cell counts (x 10^6/ml)
Day after treatment	d0	d1	d2	d3	d4
TRO in mg/ L (treatment duration in minutes)
Control	0.32	1.22	2.71	3.65	5.10
0.05 (0.5)	0.32	1.16	2.82	3.74	5.22
0.23 (1)	0.32	1.03*	2.63	3.40	4.96
0.34 (1.5)	0.32	0.71*	2.27*	3.22*	4.85
0.50 (2)	0.32	0.52*	1.95*	2.95*	4.45*
0.90 (3)	0.32	0.0.22*	1.01*	1.95*	3.47*
Pooled Standard Error		0.047	0.110	0.095	0.121

 

 

Table 3: Cell counts of Chaetoceros gracilis (x10⁶cells/mL) exposed to varying levels of ozone. Values represent mean (of four replicates. Values within a column having a * are significantly different from the control, using the Dunnett's t-test (P < 0.05).

Missing values (-) were below detectable limits.

Chaetoceros gracilis
	Cell counts (x 10^6/ml)
Day after treatment	d0	d1	d2	d3	d4	d5
TRO in mg/ L (treatment duration in minutes)
Control	0.31	1.14	3.74	4.54	5.41	5.77
0.06 (0.5)	0.31	0.51*	1.76*	4.10	5.12	5.91
0.21 (1)	0.31	0.04*	0.50*	2.52*	4.26	4.65
0.31 (1.5)	0.31	0.01*	0.11*	0.89*	3.50*	4.71
0.56 (2)	0.31	-	-	0.13*	1.55*	3.32*
0.92 (3)	0.31	-	-	-	0.38*	2.03*
Pooled Standard Error		0.048	0.125	0.356	0.347	0.369

 

Validity criteria fulfilled:

not applicable

Remarks:

no guideline study

Conclusions:

The effects of ozone exposure varied among the different species of algae, with C. gracilis appearing the most sensitive and I. galbana the most resistant. Reinocculation showed no residual effects of the treatment. The treatment was limited to an exposure to total residual oxidants varying from species to species, with an approximate range of 0.5 to 3 min. The most sensitive LOEC was determined with 0.06 TRO/L for 0.5 min of treatment, measured one day after exposition for C. gracilis.

Executive summary:

The effects of ozone treatment on three unicellular algal species, Isochris galbana, Nannochloropsis oculata and Chaetoceros gracilis were studied. I. galbana and N. oculata were found to be resistant to total residual oxidants (TRO), while C. gracilis was relatively sensitive to TRO. I. galbana remained in log-phase growth after treatment with 0.05 to 0.50 mg/L and N. oculata exhibited strong growth after exposure to TRO levels up to 0.67 mg/L. TRO levels of 0.92 and 0.90 mg/L reduced cell counts of N. oculata and I.galbana. C. gracilis initialy showed reductions of cell count at TRO levels above 0.06 mg/L. Log-phase growth was resumed 2d after exposure to TRO as high as 0.31 mg/L.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

unsuitable test system

Qualifier:

no guideline followed

Principles of method if other than guideline:

Samples of swamp water were ozonized for one-half hour and then examined microscopically for percentage removal of living organisms and organic matter.

GLP compliance:

no

Analytical monitoring:

no

Details on sampling:

n/a

Vehicle:

no

Details on test solutions:

n/a

Test organisms (species):

other: see details on test organisms

Details on test organisms:

Diatomaceae, Cyanophyceae, Chlorophyceae, Fungi, Protozoa, Rotifera, Crustacea

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 min

Post exposure observation period:

0 and 24 hours after treatment samples were examined.

Hardness:

no data

Test temperature:

no data

pH:

no data

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

Nominal: air with a low ozone concentration (0.2 to 0.3 g per cubic meter) was bubbled through the water sample at a rate of 1 L/min.

Details on test conditions:

Ozone containing air was drawn at a rate of 1 L/min through a bottle containing 0.5 L of swamp water. The percentage removal of living organisms and organic matter was examined microscopically.

Reference substance (positive control):

no

Duration:

30 min

Dose descriptor:

other: percentage of mortality

Effect conc.:

other: 0.2 to 0.3 g per cubic meter) lead into the sample water for 30 min with a rate of 1 L/min

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

other: removal of organisms

Details on results:

Average percentage removal of organisms immediately after treatment:
Diatomaceae 48.0%
Cyanophyceae 55.0%
Chlorophyceae 64.0%
Fungi 46.2%
Protozoa 41.5%
Rotifera 32.8%
Crustacea 30.0%

Validity criteria fulfilled:

not applicable

Remarks:

no guideline study

Conclusions:

Ozone affects a broad range of aquatic organisms amongst which are Cyanophycea and Chlorophycea. No observations of effects on algae after 24 hours are reported. Insufficient data are presented to draw firm conclusions.

Executive summary:

Certain forms of algae are very readily removed from water by direct oxidation while certain other forms are entirely unaffected even with protracted periods of contact with the ozone. In addition to this it has been noted that there is an increase in the odor arising from direct ozonization of algae that can only be removed after complete oxidation of the organic content of the water.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

Ozone is unstable and evaporates from the water phase, hence a flow-through study design would be necessary to maintain stable ozone test concentrations. However, the standard testing methods for algae are limited to static designs (OECD guideline 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test). A flow-through test design for algal toxicity tests has not yet been developed to date. Therefore, a full-scale guideline compliant algal growth inhibition test (72 h exposure) appears to be technically infeasible. In view of the very short half-life of ozone, the existing supportive studies (although of reliability), along with the fish toxicity data, are considered to be sufficient for ecotoxicological hazard characterisation of ozone.

Description of key information

Ozone is unlikely to be found in the water phase, as it evaporates immediately from the water phase in to the air or it reacts with other molecules, hence being not stable in water. Therefore, a flow-through study design would be necessary to keep up a defined concentration of ozone. Standard testing methods for algae are limited to static designs. 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. Therefore, additional testing of toxicity to aquatic algae or cyanobacteria would not be of added value. Available data from non-standardized tests in scientific publications are presented, showing that ozone at concentrations of 60 µg/L results in significant toxicity after an exposure for between seconds to minutes. A classification to category 1 for acute and chronic aquatic toxicity is reasonable.

Due to the unstable characteristics of ozone, additional testing of toxicity to aquatic algae or cyanobacteria would not be of added value. Available data from non-standardized tests in scientific publications are presented, showing that ozone in concentrations in 60 µg/L already displays significant toxicity after exposure in the range of seconds to minutes. A classification to category 1 for acute and chronic aquatic toxicity is reasonable.

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