Diphosphoric acid, compd. with piperazine (1:1)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 March 2003 to 27 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

June 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Official Journal of the European Communities, L 383, 179 - 186, 29 December 1992

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

At the start of the growth inhibition test, single 50 ml samples were taken from the media (without algae) containing nominal test substance concentrations of 11, 36 and 113 mg/l tested, together with a control. At the end of the test, single 50 ml samples were taken from the media containing the same test substance concentrations with algae.
The samples were kept in the freezer (< -18°C) pending chemical analysis. After receipt of the samples on 16 July 2003 they were stored at < -18°C and analysed on 24 July 2003. After analysis all samples were stored in the freezer (< -18°C). On request of the study director re-analysis of two samples occurred on 21 and on 27 August 2003.

Vehicle:

no

Details on test solutions:

The following solutions of the test substance were made:
For the range-finding test, an amount of 51.4 mg was dissolved in 500 ml of algal medium. Dilutions were then prepared in medium so as to yield a test substance concentration series of 0, 0.01, 0.10, 1.0, 10.2 and 102 mg/l
For the growth inhibition test, an amount of 114.4 mg was dissolved in 1000 ml of algal medium using a few minutes of ultrasonic treatment. The pH value of this stock solution was found to be 5.8 and was not adjusted. Dilutions were then prepared in medium so as to yield a nominal test substance concentration series of 0, 11, 20, 36, 63 and 113 mg/l with a seperation factor of 1.7.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricomutum
- Strain: CCAP 278/4
- Source (laboratory, culture collection): CCAP, The Freshwater Biological Association, the Ferry House, Far Sawrey, Ambleside, Cumbria LA22 OLP, England

A pre-culture of algae in the exponential growth phase was prepared as detailed in OECD Guideline no. 201, using the medium described below.
Test medium: The OECD medium was prepared from concentrated stock solutions in ultra pure water. It was sterilized by micropore filtration and contained 150 mg/l NaHC03 (not 50 mg/l as specified in the OECD Guideline, this in order to improve the buffer capacity of the medium). Furthermore, the medium contained Fe-citrate, because the growth of the algae can become erratic in the absence of complexed iron.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

71 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

24.2 mg CaCO3/L

Test temperature:

22.3 °C - 23.3 °C (mean 23.1 °C)

pH:

6.4 - 7.7
The pH of the media with the highest concentrations of the test substance were initially lower with the test substance concentration, however, at the end of the test the pH was found to be normalized to pH 7.3- 7.7. The pH in the control cultures were found to have remained stable during the test.

Nominal and measured concentrations:

Nominal test concentrations: 0, 11, 20, 36, 63 and 113 mg/l;
The measured concentrations ranged from 76 till 122 % of the nominal concentrations. The average mean value at the start of the test appeared to be 103 % and at the end of the test 116 %. The concentrations decreased by < 20 % as defined by the EU C.3 Guideline; the test substance appeared to be stable during the test, therefore nominal concentrations were used to report the test results. For details see attachment “T-1073FM – results growth inhibition algae.pdf”.

Details on test conditions:

TEST SYSTEM
- Test vessel: 200 ml conical test flasks covered with silicone sponge caps, autoclaved
- Fill volume: 100 ml
- Aeration: continuous shaken (Gallenkamp orbital shaker at approximately 100 rpm)
- Initial cells density: 0.8 x 10E4 cells/ml
- Control end cells density: 63.3 x 10E4 cells/ml
- No. of vessels per concentration (replicates): 3 plus 1 without algae
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Medium: OECD TG 201 medium, contained 150 mg/l NaHCO3 (not 50 mg/l as specified in the OECD Guideline, in order to improve the buffer capacity of the medium) and 80 µg/l Fe-citrate 3H2O.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultra-pure water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured at the start (medium without algae) and after 71 h in all cultures. The morphology of the algae was examined visually with the aid of a microscope at the start and end of the test. The light intensity at two different culture positions was measured at the start of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: fluorescent lamps within the standard range of 60 - 120 µmol/(s·mE2)(Bottemanne Weather Instruments Photosynthetic Radiometer RA200 Q)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Algal densities (cells/ml) and algal biovolume (μmE3/ml) were determined after 24.0, 47.5 and 71.0 h with the electronic particle counter (Coulter Multisizer IIe). Measured values were corrected for the background values in the appropriate blanks.
- Morphology: examined visually with the aid of a microscope at the start and end of the test.

RANGE FINDING STUDY
- Test concentrations: 0, 0.01, 0.10, 1.0, 10.2 and 102 mg/l (in duplicate with 4 controls)
- Results used to determine the conditions for the definitive study: effects could be expected at nominal test substance concentrations > 10 mg/l.

Reference substance (positive control):

yes

Remarks:

K2Cr207 and/or 3,5- dichlorophenol (yearly basis)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

93 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit (84.1 - 103 mg/l)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Details on results:

Test conditions: The light intensity was measured to be 83 - 99 μmol/s/m-2, being within the required limits of the Guidelines.

The EbC50 was determined to be 55 mg/l, with a 95% confidence interval of 36 - 63 mg/l.

- Exponential growth of the control: yes
- Control observations: Microscopic inspection of the morphology of algal cells in the pre-culture at the start of the test revealed no abnormal cells. At the end of the test, no abnormal cells were observed in the cultures containing different concentrations of T-1063FM.

For details on results, see attachment “T-1063FM - results growth inhibition algae.pdf”.

Results with reference substance (positive control):

Results not specified in the study report.

Reported statistics and error estimates:

In this study the EC values with respect to the growth rate and exponential growth (ErCvalues), were calculated by means of a parametric model developed by Kooijmanet al. assuming an error proportional to the number of cells.
The parametric model allows curve fitting with different assumptions about the growth pattern (exponential or logistic) and the nature of the effect of the test substance (growth rate, yield or viability of the inoculum). The best fitting model is chosen on the basis of the variance and the AIC (Akaike Information Criterion).
This calculation method is based on the assumptions made in the OECD Guideline 201.
It has been used in ring tests of algal growth inhibition test Guidelines. These ring tests have demonstrated that ErC50 values calculated by this method are identical to those calculated by the method given in the Guidelines.
EC values with respect to the area under the growth curve (EbC values) were calculated by the method given in the OECD Guideline. The values were calculated by linear interpolation of a plot of the percentage reduction in growth (IA) against the log concentration of the test substance.
Determination of the NOEC and NEC values
NOEC: The NOEC (no-observed-effect concentration) was determined as the highest concentration at which no (statistically) significant inhibition was observed. Statisticalsignificance was determined with a one tailed t-test (α=5%).
NEC: In addition, model calculations were carried out using the DEBtox software packageaccording to the Dynamic Energy Budgets Theory developed by Kooijman and Bedaux. Model parameters for population growth and their asymptotic standard deviation and correlation coefficients were estimated. The NEC (no-effect concentration), was calculated from the Profile Ln Likelihood function. This method offers a completely different basis for calculating No Effect and offers complementary information to the traditional statistical NOEC or EC 10/20 approach.

Validity criteria fulfilled:

yes

Description of key information

Effect values for Pseudokirchneriella subcapitata:

ErC₅₀ (72 hours) 93 mg/l, NOEC: 36 mg/l (EU Method C.3, OECD guideline 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

93 mg/L

EC10 or NOEC for freshwater algae:

36 mg/L