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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
other: evidence based on degradation product
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details. The short term toxicity study with the degradation product is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
The algicidal properties of the test substance in Oscillatoria rubescens were evaluated at different concentrations between 0 and 7 ppm
GLP compliance:
no
Vehicle:
no
Details on test solutions:
From a 3.5 g/L solution prepared by extemporaneous dilution of the 35% (w/w) commercial product in deionized water, appropriate amounts were used to obtain desired final concentrations expressed in ppm of the test substance.

Test organisms (species):
other: O. rubeseens D.C. and P. morum Borg.
Details on test organisms:
Test organisms. O. rubeseens D.C. and P. morum Borg. were supplied from axenic continuous cultures grown in separate 2.241. stocking cyclostats, under identical conditions:
- culture medium = 50% ASMj (Gorham et al., 1964), pH increased to 8.0 by the addition of Na2CO3 (40 mg/L) after autoclaving;
flow rate = 19 mL/h;
temperature = 20°C;
illumination = 9 W m -2 (1.500 Ix)
light/dark cycle: 18/6 h.

Total exposure duration:
30 d
Test temperature:
20°C
pH:
increased to 8.0 by the addition of Na2CO3
Details on test conditions:
In order to avoid any interference from bacteria or fungi growth, all the experiments were carried out under strict axenic conditions and only results of experiments exhibiting a total axeny are presented.

Batch cultures experiments

50 ml aliquots were drawn from the stocking cyclostat and introduced in culture tubes with the necessary amounts of H202 solution to obtain final concentrations of 0, 0.35, 0.70, 1.05, 1.40, 1.75, 3.50, 5.25 and 7.00 ppm for series A and 0, 1.40, 1.75, 2.62, 3.50 and 5.25 ppm for series B. After stirring, the tubes were incubated at 20°C under a 9 W m-2: illumination and stirred again after 5 and 24 h. After 29 h algal growth was estimated as well as optical density, pigment concentration, axeny and growth potential. The experiments were carried out in triplicate for series A and duplicate for series B.

Continuous cultures experiments

O. rubescens and P. morum were cultivated in similar conditions in two separate 1 1. cyclostats sown from the stocking cyclostats. After 2 weeks of cultivation, growth having reached steady-state, the contents of the two cyclostats were mixed so that each contained about 6000 filaments of O. rubescens and 6000 colonies of P. morum (o.d.- 0.2). One cyclostat received the necessary amount of H202 so as to obtain a final concentration of 1.75 ppm, the other remaining as control. The two cyclostats were monitored during 2 weeks for growth and axeny.

Algal growth was estimated by microscope counting of filaments for O. rubescens and colonies for P. morum. Optical density was evaluated at 650 nm. Pigment concentration was determined on algal cells collected by filtration through Whatman GF/C fiberglass filter and preserved by freezing. Chlorophyll a and carotenoids were extracted by ultrasonic disintegration in 90% acetone followed by a 15 h contact in darkness at 4°C. After filtration through a Whatman GF/C fibreglass filter, the filtrate was analysed according to Parsons and Strickland (1963) method. Biliproteins were extracted by three successive freezing-thawing cycles in phosphate buffer (pH 7.0). After centrifugation (48,000g, 4°C, 5 h) the supernatant was analysed according to Bennett and Bogorad (1973) method. Absence of bacteria and fungi was immediately checked by direct microscopic observation and confirmed by counting colonies developed on appropriate media (Nutrient Broth, Nutrient Agar, Yeast Extract) sown from tested cultures and incubated at 28°C. Growth potential was evaluated by sowing an aliquot of each culture on two different media, Gorham's ASMI and Zehnder's Z (Staub, 1961), and observing algal growth for a period of about 1 month.
Key result
Duration:
30 d
Dose descriptor:
LOEC
Effect conc.:
1.75 other: ppm
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: pigment fragility/growth/cell number
Details on results:
Batch cultures experiments:
Concentration of pigments of O. rubescens are the only and most readily determined changes effected by the test substance.

The test substance concentration tested are without any effect on P. morum whether it concerns optical density or pigments concentration or even growth potential, in spite of the fact that P. morum is present at a lower density, which is supposed to increase its sensitivity.


Continuous culture experiments:

The test substance (1.75 ppm) totally destroys O. rubescens after few hours. Microscopic observation reveals that filaments have broken up into small pieces of 10-70 um size. This result is consistent with those of batch culture experiments where this concentration appears only as a critical value by considering that the test substance efficiency is inversely related to filament density and that continuous cultures are conducted at cell density of the same order of magnitude than naturally encountered, i.e. half that of batch cultures.
Validity criteria fulfilled:
not specified
Conclusions:
Under the study conditions, the LOEC of the substance in Oscillatoria rubescens was determined to be 1.75 ppm.
Executive summary:

A study was conducted to determine the algicidal properties of the degradation product hydrogen peroxide in Oscillatoria rubescens at different concentrations between 0 and 7 ppm. Under the study conditions, the toxicity threshold of the test substance on Oscillatoria Rubescens, was about 1.75 ppm depending on culture density. Higher concentrations of the test substance destroyed more than 90% of biliproteins and carotenoids and nearly 50% of chlorophyll, whereas they had no noticeable effect on Pandorina morum, a chlorophyte tested for comparison. Under the study conditions, the LOEC of the substance in Oscillatoria rubescens was determined to be 1.75 ppm (Barroin, 1986).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
other: evidence based on degradation product
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details. The short term toxicity study with the degradation product is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
not applicable
GLP compliance:
not specified
Analytical monitoring:
yes
Details on test solutions:
The ISO algal medium was used to prepare a range of concentrations of the test substance, which were then inoculated with exponentially growing algae to a density of 104 cells/mL. Five concentrations were tested using triplicates in two experiments: range finding test and a final test. For range finding experiment, the test substance concentrations were in between 1–1000 mg/L. For final experiment, concentration range of 0.1–30 mg/L was used.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Laboratory culture of Pseudokirchneriella subcapitata was obtained from the Norwegian Institute for Water Research, Oslo, Norway (NIVA).
Test type:
static
Total exposure duration:
72 h
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.78 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Validity criteria fulfilled:
not specified
Conclusions:
Under the study conditions, the 72 h nominal EC10 and EC50 of the substance in P. subcapitata are 1.78 and 2.9 mg/L respectively.
Executive summary:

A study was conducted to determine the short term toxicity of the degradation product hydrogen peroxide in Pseudokirchneriella subcapitata, according to the modified ISO Guideline 8692. The ISO algal medium was used to prepare a range of concentrations of the test substance, which were then inoculated with exponentially growing algae to a density of 10E+4 cells/mL. Five concentrations were tested using triplicates in two experiments: range finding test and a final test. For range finding experiment, the test substance concentrations were in between 1–1000 mg/L. For final experiment, concentration range of 0.1–30 mg/L was used. The test substance content in the test solutions was analyzed using the titanium oxide-oxalate colorimetric assay. The test substance was shown to be stable in the test media. The test substance showed clear toxicity to the algae with well-defined dose response curve. Under the study conditions, the 72 h nominal EC10 and EC50 of the substance in P. subcapitata are 1.78 and 2.9 mg/L respectively (Chhetri, 2017).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
other: evidence based on degradation product
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details. The short term toxicity study with the degradation product is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Paris Commission Guidelines for testing of chemicals and drilling muds
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Triplicate samples were removed from each prepared test solution (and control) at 0 h and from the additional flasks prepared at 24 and 48 h. At the end of the test, triplicate centrifuged (2000 rpm, 2 min) and uncentrifuged samples were removed from the test flasks.
Vehicle:
no
Details on test solutions:
0.714 g of the test substance was dispersed in 0.25 L of 0.2 μm filtered algal medium. This dispersion was diluted to produce a 10 ppm nominal stock solution. The stock solution was further diluted to produce the lower test concentrations.
Test organisms (species):
Skeletonema costatum
Details on test organisms:
Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Oban, Scotland, UK
Common name: marine diatom Skeletonema costatum (Chrysophyta)
Strain: 1077/3
Algal Growth medium (Guillard's Medium): NaNO3 (75 mg), NaHPO4 * 2 H2O (5.65 mg), 1 mL stock solution 1 (Na2EDTA - 4360 mg/L, FeCl3.6H2O - 3150 mg/L, CuSO4.5H2O - 10 mg/L, ZnSO4.7H2O - 22 mg/L, CoCl2.H2O - 10 mg/L, MnCl2.4H2O - 180 mg/L, Na2MO4.2H2O - 6 mg/L), 1 mL stock solution 2 (Cyanacobalamin - 0.5 mg/L, Thiamin HCl - 100 mg/L, Biotin - 0.5 mg/L) were made up to 1 litre with filtered natural seawater and adjusted to pH 3-4 with HCl. Sodium metasilicate, 0.3 mL stock solution 3 (NaSiO3.5H2O - 100000 mg/L) were added while stirring and the pH adjusted to 8.0 with 1 N NaOH or 1 N HCl.
Test type:
static
Water media type:
saltwater
Remarks:
seawater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable
Hardness:
no data
Test temperature:
18 - 20°C
pH:
8.1 to 8.2 (start of experiment)
8.1 to 9.0 (at 72 hours)
Dissolved oxygen:
no data
Nominal and measured concentrations:
Nominal test concentrations: 0, 0.625, 1.25, 2.5, 5 and 10 ppm; concentrations used to calculate NOEC and EC50
Measured concentrations: the analytical method was not specific for the test substance; discrepancies in the results were observed.
Details on test conditions:
The inoculum volume was calculated to yield approximately 5 x 10E+3 cells/mL Skeletonema costatum. The measured cell concentration in the control at the end of the test (after 72 hours) was 14.48E+05 cells/mL. Six test vessels (250 mL Erlenmeyer flasks) were used for the controls and three vessels per exposure group. Algal cell concentrations were measured in the inoculum and in each test flask at 24, 48 and 72 hours after the commencement of the test. The temperature was maintained at 18 to 20°C through the test. Continuous, uniform illumination was provided (400-700 nm, 6610 lux). Algal cultures were rotated orbitally at 100 rpm.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.38 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.63 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The algal cell counts and derived data revealed marked inhibition of growth at concentrations of 2.5 ppm and above. By 72 hours there were indications that the inhibitory effect was less severe than found at earlier observation points, with algae in the 2.5 ppm test substance solution actively growing and limited growth in the 5 ppm solution. This reduction in inhibitory effect is reflected in the increasing EC50 values with time, 1.69 and 2.62 ppm, and NOEC values (at a 5 % significance level), 0.63 and 1.25 ppm, for 24 and 72 hours incubation respectively.
Reported statistics and error estimates:
Daily cells counts were used to determine average specific growth rates and areas under growth curves, for each definitive test flask, over the 72 hours test period. The EC50 value was calculated from average specific growth rates and areas under growth curves by probit analysis. The NOEC was calculated using average specific growth rates and areas under growth curves for each test flask in the definitive test. Data was checked for homogeneity of variance using the Fmax test. If the assumption of homogeneity of variance was not met, logarithmic and square root transformations were performed on the data in an attempt to uniform the variability between groups. If the untransformed or transformed data satisfied the homogeneity of variance analysis, the NOEC was determined using a one-way parametric analysis of variance on the appropriate data, followed by pairwise comparisons between each treatment level of the test substance and control using Dunnett's T-test.
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 72 h EC50 and NOEC of the substance in Skeletonema costatum were determined to be 1.38 mg/L and 0.63 mg/L, respectively.
Executive summary:

A study was conducted to determine the short term toxicity of the degradation product hydrogen peroxide to aquatic algae and cyanobacteria according to Paris Commission guidelines (1990) for testing of offshore chemicals and drilling muds, in compliance with GLP. Skeletonema costatum (marine diatoms) were exposed for 72 h to the test substance at nominal concentrations of 0, 0.625, 1.25, 2.5, 5 and 10 ppm under static conditions. The algal cell counts and derived data revealed marked inhibition of growth at concentrations of 2.5 ppm and above. By 72 h there were indications that the inhibitory effect was less severe than found at earlier observation points, with algae in the 2.5 ppm solution actively growing and limited growth in the 5 ppm solution. Under the study conditions, the 72 h EC50 and NOEC of the substance in Skeletonema costatum were determined to be 1.38 mg/L and 0.63 mg/L, respectively (European Chemicals Bureau, 2003).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
other: evidence based on degradation product
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details. The short term toxicity study with the degradation product is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Effect of the substance on the cultures of Nitzschia closterium was determined over an exposure period of 72 h at concentration range of 2-8 ME-5.
GLP compliance:
no
Analytical monitoring:
yes
Test organisms (species):
Nitzschia sp.
Details on test organisms:
Algal cultures of Nitzschia closterium, (Ehrenberg) W. Smith a unicellular marine diatom (originally obtained from CSIRO Division of Fisheries Algal Culture Collection) was cultured in medium (Guillard and Ryther, 1962), but with the trace elements halved, and only 45 ug iron citrate. The cultures were illuminated on a light box (Philips TL 40 W, fluorescent white) at 21°C with a 12 h/12h light/dark cycle. The cultures were maintained axenic by using autoclaved media, and carrying out transfers in a laminar-flow cupboard.
Details on test conditions:
Growth rate experiments
Algal assays were conducted as described previously (Lumsden and Florence, 1983) in filtered (0.45 um), unsupplemented sea water collected 2 km off the coast of Sydney. Nitzschia was separated from the culture medium by centrifugation and washing, and an aliquot (0.1-0.3 ml) of the washed algal suspension was added to 50 ml of sea water in a 150 ml conical flask to give an initial cell density of 2 to 4+E4 cells per ml. The flasks were covered with a loose glass cap, and placed on the light box. Cell density was measured initially and on three subsequent days using a haemocytometer. A regression line was fitted to a plot of log (cell density/initial cell density) vs. time, and growth rate (u) in cell divisions/day was calculated from, u = 3.32 log(N/No)/t, where t is growth time (days) and N and No are the number of cells at time t and zero, respectively. Occasionally, there was a lag period in growth, and growth rate was then arbitrarily calculated from the initial and final cell densities.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
25 other: uM
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The blank concentration of the test substance in a 7-day-old-culture of Nitzschia was about 1E-8 M. During the growth experiments a substantial decrease in the concentration was observed.

Many species of algae are known to liberate the test substance during photosynthesis (Stevens et al., 1973), and it has been suggested (Florence et al., 1984), that test substance production leads to self-regulation of algal growth. The lowered cell concentration of toxic test substance then leads to increased growth.

The results of this study show that the test subsance in the growth medium (unsupplemented sea water) is highly toxic to Nitzschia, even though the concentration of it decreases rapidly with time. An initial concentration of 2.5E-5 M test substance caused a 50% decrease in growth rate.

The test substance can be produced extracellularly by the oxidation of many organic compounds (e.g. phenols, amines, aminophenols) by dissolved oxygen in the growth medium (Florence, 1984), and intracellularly by the mixed function oxidase (cytochrome P450) system, or by a variety of redox compounds which divert electron flow during respiration (Hassan and Fridovich, 1979). Any agent (e.g., Cu2+ or phenanthroline-type ligands) which inactivates catalase will also increase the cellular concentration of the test substance.

 Initail test substance concentration (M x E-5)  Algal growth rate (72 h) (% of control)
 8.0  negative
 6.0  negative
 5.0  negative
 4.0  21
 3.0  40
 2.0  69
Validity criteria fulfilled:
not specified
Conclusions:
Under the study conditions, the 72 h EC50 of the substance in Nitzschia closterium was determined to be 2.5 E-5 M.
Executive summary:

A study was conducted to determine the short term toxicity of the degradation product hydrogen peroxide to the algae Nitzschia closterium. The assay was performed in filtered unsupplemented sea water. The algae culture was separated from the culture medium by centrifugation and washing, and an aliquot of the washed algal suspension was added to 50 mL of sea water in conical flask to give an initial cell density of 2 to 4 +E4 cells per mL. The flasks were covered with a loose glass cap, and placed on the light box. Cell density was measured initially and on three subsequent days using a haemocytometer. The test substance was determined spectrophotometrically by the titanium method. The results of this study show that the substance was highly toxic to Nitzschia, even though the concentration of the test substance decreases rapidly with time. Under the study conditions, the 72 h EC50 of the substance in Nitzschia closterium was determined to be 2.5 E-5 M (Florence, 1986).

Description of key information

Under normal use conditions, potassium superoxide is not anticipated to be exposed to the aquatic environment. Exposure to the water is only possible if KO2 is accidentally released. However, when exposed, potassium superoxide reacts rapidly with water to produce potassium hydroxide (KOH), oxygen (O2) and potassium hydrogen peroxide (KHO2), which slowly degrades to KOH, H2O2 (hydrogen peroxide) and O2. Therefore, aquatic toxicity of KO2 could be anticipated to be due, in part, to the degradation product H2O2. KOH further dissociates into potassium and hydroxyl ions which constitute normal ion pool of the medium. On the other hand, hydrogen peroxide is likely to degrade within a short time in aquatic medium due to many alternative and competitive degradation pathways. As a result, the contribution of toxicity from hydrogen peroxide is likely to be low. Overall, the aquatic toxicity of potassium superoxide is likely to be low and a quantitative estimation of the hazard potential will not be appropriate due to its rapidly changing degradation kinetics.

Key value for chemical safety assessment

Additional information

A study was conducted to determine the short term toxicity of the degradation product hydrogen peroxide to aquatic algae and cyanobacteria according to Paris Commission guidelines (1990) for testing of offshore chemicals and drilling muds, in compliance with GLP. Skeletonema costatum (marine diatoms) were exposed for 72 h to the test substance at nominal concentrations of 0, 0.625, 1.25, 2.5, 5 and 10 ppm under static conditions. The algal cell counts and derived data revealed marked inhibition of growth at concentrations of 2.5 ppm and above. By 72 h there were indications that the inhibitory effect was less severe than found at earlier observation points, with algae in the 2.5 ppm solution actively growing and limited growth in the 5 ppm solution. Under the study conditions, the 72 h EC50 and NOEC of the substance in Skeletonema costatum were determined to be 1.38 mg/L and 0.63 mg/L, respectively (European Chemicals Bureau, 2003).

A study was conducted to determine the algicidal properties of the degradation product hydrogen peroxide in Oscillatoria rubescens at different concentrations between 0 and 7 ppm. Under the study conditions, the toxicity threshold of the test substance on Oscillatoria Rubescens, was about 1.75 ppm depending on culture density. Higher concentrations of the test substance destroyed more than 90% of biliproteins and carotenoids and nearly 50% of chlorophyll, whereas they had no noticeable effect on Pandorina morum, a chlorophyte tested for comparison. Under the study conditions, the LOEC of the substance in Oscillatoria rubescens was determined to be 1.75 ppm (Barroin, 1986).

A study was conducted to determine the short term toxicity of the degradation product hydrogen peroxide to the algae Nitzschia closterium. The assay was performed in filtered unsupplemented sea water. The algae culture was separated from the culture medium by centrifugation and washing, and an aliquot of the washed algal suspension was added to 50 mL of sea water in conical flask to give an initial cell density of 2 to 4 +E4 cells per mL. The flasks were covered with a loose glass cap, and placed on the light box. Cell density was measured initially and on three subsequent days using a haemocytometer. The test substance was determined spectrophotometrically by the titanium method. The results of this study show that the substance was highly toxic to Nitzschia, even though the concentration of the test substance decreases rapidly with time.Under the study conditions, the 72 h EC50 of the substance in Nitzschia closterium was determined to be 2.5 E-5 M (Florence, 1986).

A study was conducted to determine the short term toxicity of the degradation product hydrogen peroxide in Pseudokirchneriella subcapitata, according to the modified ISO Guideline 8692. The ISO algal medium was used to prepare a range of concentrations of the test substance, which were then inoculated with exponentially growing algae to a density of 10E+4 cells/mL. Five concentrations were tested using triplicates in two experiments: range finding test and a final test. For range finding experiment, the test substance concentrations were in between 1–1000 mg/L. For final experiment, concentration range of 0.1–30 mg/L was used. The test substance content in the test solutions was analyzed using the titanium oxide-oxalate colorimetric assay. The test substance was shown to be stable in the test media. The test substance showed clear toxicity to the algae with well-defined dose response curve.Under the study conditions, the 72 h nominal EC10 and EC50 of the substance in P. subcapitata are 1.78 and 2.9 mg/L respectively(Chhetri, 2017).