Tetrakis(hydroxymethyl)phosphonium chloride

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant study, performed according to ISO method 10253; but no data is avalaible on the sample: purity, batch number, etc..

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

124-64-1 as marketed

IUPAC Name:

124-64-1 as marketed

Sampling and analysis

Analytical monitoring:

not specified

Details on sampling:

- Concentrations:
The test solution concentrations used were 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L (as test item) together with a culture medium control.
A stock solution of 100 mg/L of test item was prepared by dissolving approximately 1.0 g of the test material in 1 L of culture medium. Varying aliquots of this stock were then added to volumes of sterile test medium whilst stirring. The control consisted of culture medium only.
After the preparation, a visual assessment of the test solutions showed all the test solutions to be clear and colourless.
The test vessels were borosilicate glass conical flasks of 250 mL nominal capacity closed with polyurethane foam bungs. Each test replicate flask contained 100 mL of the test solution. Using aseptic techniques, volumes of the appropriate test solution were dispensed to each test and blank vessel, and the remaining test solutions used for physical analyses.

- Sampling method:
Six replicate cultures of the culture medium control and triplicate cultures of each test material concentration were employed. The positions of the vessels in the incubator were randomised and re-randomised, daily, by rows. One blank vessel (without algal inoculum) was incubated concurrently for the control and each test concentration.

- Sample storage conditions before analysis:
The cultures were incubated at 20 ± 2°C (the nominal temperature), under continuous "cool white" illumination, with orbital shaking at approximatel 100 rpm (Gallenkamp type INR-401 orbital incubator).

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: Skeletonema costatum
- Strain: CCAP 1077/1C
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, PO Box 3, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): no precised data. A culture of the alga in the exponential growth phase was used as inoculum.
- Method of cultivation: the culture was grown in the medium, and under the environmental conditions described in the ISO standard.

- Inoculum:
A culture of the alga in the exponential growth phase was used as inoculum.
Each replicate test vessel was inoculated with 385 µL of the inoculum culture to give a nominal particle density of 0.150E+4 particles/mL. This was considered to be the equivalent of 0.300E+4 cells/mL, since the average number of individual algal cells per growth filament is approximately two. Three 100 mL volumes of Coulter electrolyte, inoculated in the same manner, had a mean measured particle density of 0.155E+4 particles/mL. This value was used for growth calculations.

ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: not examined

Study design

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Test temperature:

The temperature of the incubator was measured daily by a thermometer calibrated to 0.1°C and conforming to BS593, and was continuously monitored, with hourly recording of values, using an automatic recording system linked to a thermistor.

pH:

The pH of each test solution was measured at the start of the test, using the excess of remaining after filling the test vessels. At the end of the test, the pH of two of the replicate test solutions (containing algae) from the control and each each test concentration was determined. The measurements were made using a Corning Model 240 pH meter.

Salinity:

The salinity of excess test solution medium was measured at the start of the test using a Kent Instrument model MC5 salinometer.

Nominal and measured concentrations:

The algal particle densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter model ZB, counting at a lower threshold equivalent spherical diameter of approximately 2.9 µm.
After 1, 2 and 3 days (24, 48 and 72 hours), samples were removed from each test and blank vessel. The appropriate blank particle count was substractred from that of the test culture to obtain the particle density.

Details on test conditions:

TEST SYSTEM
- Test vessel: borosilicate glass conical flasks of 250 mL nominal capacity
- Type: closed with polyurethane foam bungs
- Material, size, headspace, fill volume: each test replicate flask contained 100 mL of test solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through
- Renewal rate of test solution (frequency/flow rate): no renewal of the test solution
- Initial cells density: 3000 algae cells/mL
- Control end cells density:
- No. of organisms per vessel: 3000 algae cells/mL * 100 mL = 300000 algae cells
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 0


GROWTH MEDIUM
- Standard medium used: yes (reconstituted test water according to the test guideline)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
1. To 900 mL of seawater, filtered to a least 1 µm, add:
(a) 15 mL of solution A
(b) 1 mL of solution B
(c) 0.5 mL of solution C
(d) distilled water to 1L

2. Filter sterilise complete medium into sterile vessels
3. Adjust pH to 8.0 ± 0.2 with NaOH or HCL
4. Store solutions A, B and C under refrigeration.

Solution A:
FeCl3.6H2O 0,048 g
H3BO3 1,140 g
Na2EDTA.2H2O 1,0 g
MnCl2.4H2O 0,144 g
ZnSO4.7H2O 0,045 g
CoCl2.6H2O 0,404 mg
CuSO4.5H2O 0,157 mg
Distilled H2O to 1L

Solution B:
K3PO4 1,5 g
NaNO3 25,0 g
Na2SiO3.9H2O 10,0 g
Distilled H2O to 500 mL

Solution C:
Thiamin hydrochloride 50 mg
Biotin 0,01 mg
Vitamin B12 0,1 mg
Distilled H2O to 1 L

- Culture medium different from test medium: no


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, for the preparation of the culture medium. No after.
- Photoperiod: continuous illumination
- Light intensity and quality: the light intensity, measured once during the study, was 7810 lux (by cosine receptor)
- Salinity (for marine algae): the salinity of excess test culture medium measured at the start of the test was 31.4‰.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: by an electronic particle counter, using a Coulter counter model ZB, counting at a lower threshold equvalent spherical diameter of approximately 2.9 µm.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: No
- Test concentrations: -
- Results used to determine the conditions for the definitive study: no data

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.025 other: mg/L THPC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL could not be determined

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.05 other: mg/L THPC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL could not be determined

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.12 other: mg/L THPC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.054 to 0.368 mg/L THPC

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.025 other: mg/L THPC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL could not be determined

Effect concentrations 5

Duration:

72 h

Dose descriptor:

other: Lowest significant effect concentration

Effect conc.:

0.05 other: mg/L THPC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL could not be determined

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.424 other: mg/L THPC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.136 to > 0.8 mg/L THPC

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- 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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The control cell density shall have increased by a factor of 38 (that is more than 16) in 72 h. The variation coefficient of the control specific growth rates does not exceed 7 % (it reached 0.785). The control pH shave not increase by more than 1.0.

Conclusions:

THPC inhibited the growth of the marine algae Skeletonema costatum with EC50 and NOEC values for the test parameter « growth rate » of 0.424 mg/L and 0.025 mg/L, respectively. The EC50 and NOEC values for the test parameter « biomass » were calculated as 0.12 mg/L and 0.025 mg/L, respectively.
Therefore, THPC should be considered as "very toxic to the aquatic organisms tested".

Executive summary:

The influence of the test substance THPC on the growth of the marine algae Skeletonema costatum was investigated in a 72 hour static test according to the ISO Guideline No.10253 and the GLP.

The nominal concentrations of the test item of 0.0078, 0.0156, 0.0313, 0.0625, 0.125,0.25,0.50 and 1.0 mg/L were tested in parallel with a control.

The concentrations of the test substance in the test media were not measured, so not compared to the nominal concentrations.The biological results were related to the mean nominal concentrations values of THPC.

THPC had a statistically significant inhibitory effect on the algal particle density after the test period of 72 hours at the concentration of 0.05 mg/L and at all higher test concentrations. The average growth rate was significantly lower than the control at the same concentration.

The 72 hour EC50 values for the two test parameters “growth rate” and “biomass” were determined to be 0.424mg/L and 0.12mg/L,respectively. The 72 hour NOEC was 0.025 mg/L with these both parameters.

The validity criteria requested by the ISO Guideline No.10253 were fulfilled.

Based on these results,THPC should be considered as “very toxic to the aquatic organisms tested”.