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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
endpoints: intracellular ATP and cell recovery, cell count as marker for phytotoxicity
Deviations:
yes
Remarks:
exposure time: 96h
Principles of method if other than guideline:
The unicellular green alga Pseudokirchneriella subcapitata (also known as Selenastrum capricornutum) was exposed to the test substances for 96 h in 96-well microplates. Subsequently, cell count was performed as marker of phytotoxicity.
GLP compliance:
no
Specific details on test material used for the study:
Sodium azide (NaN3) was an ACS grade Fisher Scientific chemical.
Analytical monitoring:
no
Details on sampling:
no sampling was reported
Vehicle:
no
Details on test solutions:
Sodium azide (NaN3) was an ACS grade Fisher Scientific chemical. Prior to testing, the pH of all sample working solutions was adjusted to 7. Working solutions were then filter sterilized (Gelman, GA-8; 0.20-µm filter). Filtration provided axenic conditions for algae in the ensuing intoxication experiments to eliminate potential bacterial growth. In all cases, nominal prefiltration chemical concentrations were utilized to report toxicity data.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The unicellular green alga Pseudokirchneriella subcapitata (ATCC 22662, also known as Selenastrum capricornutum) was grown in 30% AAP (algal assay procedure) medium with Na2EDTA (sodium ethylenediaminetetraacetate), under previously reported experimental conditions of temperature (24 ± 2 °C) and continuous light (cool white fluorescent lamps, 95 µE•m-2•s-1) for stock culture maintenance and all experiments (Blaise, 1986).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
24 ± 2 °C
pH:
adjusted to pH 7
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
no datas
Details on test conditions:
TEST SYSTEM
- Test vessel: 96-well sterile microplates with lid
- Type (delete if not applicable): closed
- Material : polystyrene
- Max. fill volume: 300 µL
- Aeration: none: microplate with plastic lid was sealed into a transparent polyester bag to avoid evaporation
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: ca. 910 cells per mL
- No. of organisms per vessel: 200
- No. of vessels per concentration (replicates): 5-8
- No. of vessels per control (replicates):8


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: U.S. EPA Algal assay medium, 30%


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: to 7
- Photoperiod: continuous
- Light intensity and quality: cool white fluorescent lamps, 95 µE•m-2•s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter Coulter Counter (model ZM, 70 µm cell aperture)
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL: 0.242-0.429
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
Dose—response curves were obtained by the least squares method with a nonlinear y = ax b model that best described the curvilinear relationship existing between variables for our data (as in Fig. 1). Data were arcsin transformed and a line of best fit by linear regression was applied to enhance visual presentation and interpretation (Figs. 2 and 3). Arcsin transformation is the most appropriate for proportion or percentage-type data (Zar, 1984). Additionally, t tests or covariance analyses followed by Tukey multiple comparisons were applied for testing differences among regression lines (slopes and elevations). EC50 s were determined by linear regression analysis of 3-5 points lying in the 15-85% inhibition concentration range. All regressions had correlation coefficients >90%.

ATP levels were markedly depressed, but no effects on recovery were observed, see Figure 1.

Validity criteria fulfilled:
no
Conclusions:
The EC50 of Sodium azide after 96 h exposure to green algae Selenastrum capricornutum was 0.35 mg/L.

The short-term toxicity of Sodium azide on the green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) was assessed using a 96-well microplate assay after 96 h exposure by cell counting.
The resulting EC50 was reported to be 0.35 mg/L, further information on concentrations tested or result data were not given.
Executive summary:

In a 96 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to Sodium azide under static conditions in accordance with the method described in Blaise et al. 1986.  The EC50values based on cell number was 0.348 mg/L. 

 

There were no compound related phytotoxic effects.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for acute algae toxicity study.

 

Results Synopsis

 

Test Organism: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)

Test Type (Flowthrough, Static, Static Renewal): Static

 

96 hr EC50:  0.348 mg/L                     95% C.I.: 0.242 to 0.429 mg/L

 

Endpoint(s) Effected: Cell counting (i.e. growth rate)

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
Remarks:
Well conducted study, but not described in sufficient detail and not performed according to guideline or GLP.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Giant kelp (Macrocystis pyrifera) were exposed to sodium azide in water for different times:
1) 48h continuous exposure.
2) 48h or 96h exposure, followed by a 17d or 20d recovery period, respectively.
3) 19d or 24d continuous exposure.
Measured parameters included gametophyte germination, gametophyte growth (germ-tube growth), and sporophyte numbers.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
No analytical monitoring performed.
Vehicle:
no
Details on test solutions:
No vehicle used.
Test organisms (species):
other: Macrocystis pyrifera
Details on test organisms:
TEST ORGANISM
- Common name: giant kelp
- Source (laboratory, culture collection): no data
- Age of inoculum (at test initiation): no data
- Method of cultivation:
1) 48h exposure: no data
2) exposure-recovery experiments: slides containing gametophyte cultures were transferred from azide spiked media to containers with control seawater containing nutrients necessary for gametogenesis (PES plus germanium dioxide to control diatoms). In addition, the lighting was changed from cool white fluorescent to full spectrum Vitalights (50 µEm-2sec-1) to induce gametogenesis. Test solutions were renewed after every 96h.
3) continuous exposure experiments: indentical to 2)
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
in experiment 1) and 2). Exposure duration 19 or 24d in experiment 3).
Post exposure observation period:
Experiment 2): 15 or 17days.
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
nominal concentrations: 0, 5.6, 10, 18, 32, 56, 100, 180 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: "container", no further information given
- Renewal rate of test solution (frequency/flow rate): in exposure-recovery and continuous exposure experiments: every 96h
- No. of organisms per vessel: no data
- No. of vessels per concentration (replicates): no data
- No. of vessels per control (replicates): no data
- No. of vessels per vehicle control (replicates): no data


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition: seawater, in exposure-recovery and continuous exposure experiments supplementation with nutrients (PES + germanium dioxide)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: no data
- Light intensity and quality: cool-white fluorescent light or full-spectrum Vitalight (50 µEm-2sec-1)
- Salinity (for marine algae): no data


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Other: germination, germ-tube growth, sporophyte numbers


STATISTICS:
All treatments were analyzed using ANOVA followed by Dunnett's test to determine NOECs. Concentrations causing 50% inhibition of germination and sporophyte production (EC50) were calculated using the Spearman-Karber method.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: germ tube length
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
70.7 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
other: germination
Remarks on result:
other: Average value from two independent experiments with EC50 of 51.53 and 89.9 mg/L, respectively.
Duration:
19 d
Dose descriptor:
NOEC
Effect conc.:
< 3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: sporophyte production
Duration:
19 d
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: sporophyte production
Remarks on result:
other: 95% CI: 0.9 - 1.3 mg/L
Details on results:
- Exponential growth in the control (for algal test): no data
- Any stimulation of growth found in any treatment: 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
See also Table 1 further below.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
high dataset variability

Table 1: Overview of the different ECx-values for each endpoint evaluated.

Endpoint

Azide concentration (mg/L)

NOEC

EC50

95% CI

Germination (48h exposure)

18.0

51.53

49.3 – 53.8

Germ tube length (48h exposure

18.0

 

 

Sporophyte production (19d CE)

< 3.2

1.12

0.9 – 1.3

Sporophyte production (48h/17d ER)

> 180.0

127.7

28.0 – 580.8

Germination (48h exposure)

18.0

89.9

No statistical results

Germ tube length (48h exposure

10.0

 

 

Sporophyte production (24d CE)

< 5.6

1.1

No statistical results

Sporophyte production (96h/20d ER)

5.6

9.2

8.6 – 9.8

CE: Continuous exposure

ER: exposure-recovery

Validity criteria fulfilled:
not applicable
Remarks:
Study not performed according to guideline.
Conclusions:
After short-term incubation (48 or 96h), Sodium azide inhibits germination and germ tube growth. The most sensitive NOEC was obtained after 96h exposure and was 10 mg/L. The EC50 ranged between 51.53 and 89.9 mg/L.
Executive summary:

The developmental toxicity of Sodium azide against algae was determined by incubation of giant kelp (Macrocystis pyrifera) with Sodium azide. Incubation times comprised a short-term exposure (48 or 96h), an exposure-recovery experiment (ER, 48 or 96h exposure and 17 or 20d recovery, respectively) and a 19 or 24d continuous exposure (CE). Parameters studied were germination and germ tube lenght (short term exposure) or sporophyte production (ER and CE).

Short term experiments revealed that sodium azide impairs kelp germination with an EC50 of 70 mg/L. The NOEC was determined as 18 mg/L. In long term experiments, especially at continuous exposure, the NOEC was lower, under-running the lowest concentrations tested (3.2 or 2.6 mg/L). The exposure-recovery experiment showed that gametophytes cultured in azide concentrations significantly inhibiting germination and growth for 48h are able to recover and undergo reproduction when transferred to azide-free media (EC50(48h) = 127.7 mg/L). After 96h incubation in azide containing medium, some recovery was indicated, though it is apparent that except in the lowest azide concentration, gametogenesis continued to be suppressed (EC50(96h) = 9.2 mg/L). These data suggest that the initial exposure duration determines whether kelp gametophytes exposed to sodium azide are competent to recover and successfully reproduce when transferred to uncontaminated media.

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
Remarks:
The endpoint is oxygen production of greenalge. The study itself is conducted according to national standard methods and well documented.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
other: Deutsche Einheitsverfahren zur Wasser-, Abwasser und Schlammuntersuchung DEV L 12
Principles of method if other than guideline:
- Principle of test: The photosyntheitic O2-Production of green algae is evaluated.
- Short description of test conditions: mixed culture of Chlorococcales sp is exposed to the test substance; Stock solution 1.150 g/L, dilution series, spacing factor 3; 6 test concentrations; 2 replicates and control; one bottle was exposed in the light climate chamber one in a dark climate cha,ber (20 °C both). The oxygen production of a test batch was calculated from the difference between light and dark bottle values ​​and related to the oxygen production value of the control to determine the percentage inhibition value.
- Parameters analysed / observed: oxygen production compared to the control.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Stock solution was prepared with tap water and diluted to five different concentrations.
Test organisms (species):
Chlorococcum sp.
Details on test organisms:
TEST ORGANISM
- Common name: Chlorococcales sp., mixed cultures; mainly: Scenedesmus sp.
- Age of inoculum (at test initiation): algae are harvested during logarithmic growth rate and diluted 1:23 with tap water resulting in an oxygen production of 6 +/- 2 mg/(Lx24 h)
- Method of cultivation: aerated algae culture tubes (length 50 cm, diameter, 3 cm, culture volume 200 mL); medium according to Staub et al. 1961; room temperature, daylight

ACCLIMATION
- Acclimation period: lab culture, no acclimation phase; culture conditions similar to test conditions
- Culturing media and conditions (same as test or not): according to Staub et al. 1961
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
24 h
Test temperature:
20 °C
pH:
7
Nominal and measured concentrations:
Nominal: 0.003, 0.01, 0.03, 0.1, 0.3, 1 g/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Wide-neck bottles
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: Glass, 100 mL nominal volume
- Aeration: No
- Initial cells density: No data
- Control end cells density: No data
- No. of vessels per concentration (replicates): No data
- No. of vessels per control (replicates): No data
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: No
- Detailed composition if non-standard medium was used: Algal culture medium "Z8", cf. http://www-cyanosite.bio.purdue.edu/media/table/Z8.html

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water
- Total organic carbon: No data
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/Mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: No data

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 3000 lux, Osram light colour 25

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Not determined
- Chlorophyll measurement: Not determined
- Other: Oxygen production over 24 h relative to dark controls

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: No
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
176 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: oxygen production
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: oxygen production
Validity criteria fulfilled:
not applicable
Conclusions:
The effect of 24h sodium azide incubation on algaeal photosynthetic oxygen production as surrogate marker of algaeal growth was determined.
The resulting EC10 and EC50 values were 55 and 176 mg/L, respectively.
Executive summary:

 In a 24 hour acute toxicity study, the cultures of mixed Chlorococcales (mainly Scenedesmus sp.) were exposed to Sodium azide under static conditions in accordance with the method described in Blaise et al. 1986.  The EC50 and EC10 values based on oxygen consumption was 176 mg/L and 55 mg/L, respectively. 

 

There were no compound related phytotoxic effects.

 

This toxicity study is classified as supplementary, because it is only conducted for 24 h and does not evaluate the growth rate, though in accordance with German standardized methods for evaluation of water, sewage and sludge (Deutsche Einheitsverfahren zur Wasser-, Abwasser und Schlammuntersuchung DEV L 12).

 

Results Synopsis

 

Test Organism: Chlorococcales (mainly Scenedesmus sp.)

Test Type (Flowthrough, Static, Static Renewal): Static

 

24 hr EC50:  176 mg/L       

24 hr EC10: 55 mg/L

Endpoint(s) Effected: Oxygen production

Description of key information

Three peer reviewed studies on aquatic algae are available. As key study Hickey et al. (1991) is chosen. They conducted a microplate assay with cultures of Pseudokirchneriella subcapitata which were exposed to Sodium azide under static conditions in accordance with the method described in Blaise et al. (1986), similar to conditions described in the OECD guideline 201. The 96 h EC50value based on the cell number was 0.348 mg/L and is considered as key value. The supportive study from Krebs (1991) exposed the unicellular green algae Chlorococcum sp. to Sodium azide for 24 h and measured differences in the oxygen production. Based on this endpoint the 24 h EC50derived was 176 mg/L. Another supportive study was conducted with the macrophyte Macrocystis pyrifera, exposing the brown algae for 48 h to sodium azide. The endpoints germination and germ tube length were evaluated for the exposure period of 48 hours. The determined EC50 was 70.7 mg/L for endpoint germination and a NOEC of 10 mg/L for germ tube length (most sensitive endpoints). Both supporting studies do not apply standardised methods, however, they support the key study as the derived effect concentrations are much higher, than the derived value from the key study. Therefore, it can be concluded that the EC50 from the key study is conservative enough to be used for a PNEC derivation.

Key value for chemical safety assessment

EC50 for freshwater algae:
348 µg/L

Additional information