Hydroxyl-2-pyridone

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-05-23 to 1994-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 122-2 (Algal Toxicity, Tiers I and II)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxyl-2-pyridone

EC Number:

212-506-0

EC Name:

Hydroxyl-2-pyridone

Cas Number:

822-89-9

Molecular formula:

C5H5NO2

IUPAC Name:

1-hydroxy-1,2-dihydropyridin-2-one

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): Oxy PYRION
- Physical state: solid (powder)
- Appearance: white speckled gold powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 93100020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at ambient temperature in the original container until required
- Stability under test conditions: not specify

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: the effect of oxypyrion on the growth of Selenastrum capricornutum was determined over a period of 72 hrs (OECD 201) and 120 hrs (US EPA. FIFRA Methods 122.2 and 154.10).
Samples for possible analysis were taken from all test concentrations and the control at t= 0, t= 72h and t= 120h. 20 mL of volume was taken from the approximate centre of the test vessels. At the end of the exposure period, the replicates were pooled at each concentration before sampling
- Sample storage conditions before analysis: Additionally, reserve samples of 20 mL were taken for possible analysis. The samples were stored in a refrigerator.
The samples of aqueous test media (20 mL) were diluted with HPLC mobile phase within the concentration range of 0.15 to 0.5 mg/L. The concentrations of Oxypyrion in these samples were then determined by high performance liquid chromatography (HPLC) using a spectrophotometric detector

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: at the start, an aliquot (40 mL) of the inoculum was added to each of the prepared test vessels to give a density of 1x10^4 cells/mL. At 10 mg/L, the test medium was made from a concentrated aqueous dilution (nominally 100 mg/L) in which test material has been added directly to mineral salts medium. At 5 mg/l and below the test media were prepared from a series of intermediate stock dilutions (3.13 to 50 mg/L) prepared in mineral salts medium from the concentrated aqueous stock. An aliquot (10 mL) of the appropriate stock dilution was added to each vessel.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green algae
- Strain: Selenastrum capricornutum (Pseudokirchneriella subcapitata)
- Source (laboratory, culture collection): Culture Centre for Algae and Protozoa (CCAP), Freshwater Biological Association, Ferry House, Far Sawrey, Ambleside, Cumbria
- Method of cultivation: liquid cultures for the test inoculum were established by inoculation of the appropriate algal medium (50 mL) with cells aseptically removed from slope cultures. Subsequently, subcultures were prepared from these liquid cultures by the aseptic transfer of aliquots into fresh algal medium, which were then incubated for several days, until the required cell density had been achieved. Liquid cultures were maintained in a temperature-controlled illuminated orbital incubator, at 20 - 25°C, at a nominal shaking speed of 175 revolutions per minute.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): In the 7 hr and 120-hr tests, the test inocula containing 2.5 x 10^4 cells/mL were prepared in the appropriate sterile algal megium, by the dilution ot liquid subcultures of 1.3 x 10^6 cells/mL and 5.8 x 10^5 cells/mL respectively.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Remarks on exposure duration:

the effect of oxypyrion on the growth of Seleastrum capricornutum was determined over a period of 72 hrs (OECD 201) and 120 hrs (US EPA. FIFRA Methods 122.2 and 154.10).

Test conditions

Hardness:

not reported

Test temperature:

72h test
At t= 0h: 22.0-22.4
At t= 72h: 22.7-23.3

120h test
At t= 0h: 22.7-22.9
At t= 120h: 25.0-25.6

pH:

72h test
At t= 0h: 7.23-7.97
At t= 72h: 7.78-9.75

120h test
At t= 0h: 7.17-7.31
At t= 120h: 6.98-7.49

Dissolved oxygen:

not reported

Salinity:

not relevant

Conductivity:

not relevant

Nominal and measured concentrations:

72h final test (OECD 201)
Nominal concentrations: 0, 0.313*, 3.13**, 0.625*, 6.25**, 1.25*, 12.5**, 2.5, 5, 10 mg/L
Measured concentrations (mg/L) at t=0h (2 replicates per conc.): n.d., 0.125* (0.114*); 2.82** ( 2.89**); 0.344* (0.329*); 5.91** (5.78**); 0.944* (0.925*);
12.0** (11.9**); 2.39 (2.35); 4.57 (4.51); 10.3 (10.3)
Measured concentrations (mg/L) at t=72h (2 replicates per conc.): n.d., 0.185 (0.187), 0.481 (0.448), 0.977 (1.06), 2.18 (2.25), 4.62 (4.81), 9.69 (9.59)
Mean values (mg/L): 0.286, 0.585, 1.20, 2.29, 4.63, 9.97
Chemical analysis indicated that intended exposure concentrations were achieved and adequately maintained.

120h final test (FIFRA)
Nominal concentrations: 0, 0.313*, 3.13**, 0.625*, 6.25**, 1.25, 2.5, 5, 10 mg/L
Measured concentrations (mg/L) at t=0h (2 replicated per conc.): n.d., 0.219* (0.226*), 3.09** (3.08**), 0.567* (0.517*), 6.75** (6.54**),1.19* (1.18*), 2.49 (2.64), 4.98 (5.35), 10.9 (10.8)
Measured concentrations (mg/L) at t=72h (2 replicates per conc.): n.d., 0.117 (0.116), 0.307 (0.298), 0.772 (0.761), 1.79 (1.84), 4.24 (4.31), 8.86 (9.04)
Mean values (mg/L): 0.309, 0.665, 0.976, 2.19, 4.72, 9.90
Chemical analysis indicated that intended exposure concentrations had fallen to between 65 and 83% of their initial values.


* measured concentrations at these levels were below the LOQ and have not been used in the calculation of the mean measured values.
** measured concentration in aqueous stock media which were used in the calculation of the test concentrations (**x 0.1)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): conical
- Material, size, headspace, fill volume: 250 mL all-glass flasks filled with 50 mL test solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: ??
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 7

GROWTH MEDIUM
- Standard medium used: yes (OECD 201 and US EPA mineral salts medium)

TEST MEDIUM / WATER PARAMETERS:
- Source/preparation of dilution water: concentrated solutions of the mineral salts were prepared in filtered dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification.
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature measured continuously, pH at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: the test vessels were sterilsed bij autoclaving (120°C for 15 minutes)
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: either seven (72-hour exposure) and six (120-hour exposure) horizontally-mounted, three-foot 30 watt "cool-white" fluorescent tubes. The light intensity at the position occupied by culture flasks during tests was estimated to be approximately 11000 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] The number of cells in each sample was determined using
a haemocytometer (Improved Neubauer); estimates of cell numbers were based on the means of four or eight individual cell counts.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10

- Range finding study
- Test concentrations: 1.00, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL 95% (5.55-8.24 mg/L)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

7.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL 95% (6.67 and 7.41 mg/L)

Effect concentrations 4

Duration:

120 h

Dose descriptor:

NOEC

Effect conc.:

4.72 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: in the 120 hr test at 1.25 and 2.5 mg/L resp. measured concentrations had decreased to 65 and 71% suggesting that the test material was not stable under the conditions of the test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Selenastrum capricornutum (Pseudokirchneriella subcapitata) was performed with the test substance Oxypyrion according to the OECD guideline 201 (GLP conditions) and a 120-h growth inhibition test according the US EPA.FIFRA Methods 122.2 and 154.10.
Under the conditions of the OECD procedure 201 (72-hours) exposure to Oxypyrion caused a significant reduction in both the average specific growth rates and biomass of Selenastrum capricornutum cultures at levels of 2.29 mg/L and above. The 72-h EC50 for growth rate inhibition was 6.60 mg/L (95% confidence limits 5.55 and 8.24 mg/L).
Under the conditions of the US EPA.FIFRA Methods 122.2 and 154.10 (120-hours) exposure to Oxypyrion caused a significant reduction in both the average specific growth rates and biomass of Selenastrum capricornutum cultures at levels of 9.90 mg/L and above. The 120-h EC50 for growth rate inhibition was 7.02 mg/L (95% confidence limits 6.67 and 7.41 mg/L). The results of the test can be considered reliable without restrictions.

The slight reduction in toxicity of Oxypyrion observed under the conditions of the US EPA.FIFRA test compared to that obtained in the OECD test conditions was considered to be due to the instability of the test material over the longer exposure period (120 hrs).