Amidinourea phosphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Remarks:

Pseudokirchneriella subcapitata Hindák

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jun 2017 – 31 Aug 2017

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:

2006, corrected 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16VL8189
- Expiration date of the lot/batch: 04 Feb 2018
- Purity test date: 05 Aug 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (=< 30 deg C), dark, dry

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No

Analytical monitoring:

yes

Details on sampling:

The analytical verification of test item concentrations in test solutions was done by analysing the content of guanylurea phosphate in the samples during the test. The analysis of samples was performed in the analytical laboratories of the test facility with a suitable analytical method. The results of the analysis are part of the raw data and this final report. The content of the analyte in the test solution samples was determined by analysing with HPLCMS/MS. The analytical method was validated with regard to specificity, linearity, accuracy (recovery), precision and limit of quantification. Validation was performed in accordance with SANCO/3029/99 rev. 4 from 11/07/2000.
Analytical samples of all test item concentrations and control were analysed at test start (0 hours fresh) and at test end (72 hours aged).

Vehicle:

no

Details on test solutions:

The necessary amount of test item for preparing the stock solution S1 was weighed on a weighing scoop and transferred to a volumetric flask. Test medium (see Appendix B) was added up to the bench mark and the solution was homogenised by shaking and treated with 5 minutes of ultrasonication. Afterwards the solution was clear and transparent. Lower test solutions were prepared by dilution of the stock solution S1 with test medium. Algae were added to each solution individually resulting in nominal cell densities of 0.5 × 104 cells per mL in each solution.

Test organisms (species):

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

Details on test organisms:

The test organism Pseudokirchneriella subcapitata Hindák (Sphaeropleales: Selenastraceae), strain: SAG 61.81, was purchased from a commercial supplier, e.g. MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany.
The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Culture conditions are as follows:
Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 – 120 µEm-2s-1)
Temperature: 21 - 24 °C x Culture flasks: 100 mL Erlenmeyer flasks
CO2 supply by shaking on a rotating shaker, approximately 105 rpm Cells from this semi-continuous liquid stock culture were used for the test. 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

Not applicable

Hardness:

Not measured

Test temperature:

22.6 – 23.0 °C

pH:

7.29 to 8.33

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Conductivity:

Not measured

Nominal and measured concentrations:

The following nominal test item concentrations were tested (spaced by a factor of 3.2): 100, 31.3, 9.77, 3.05, 0.954 mg/L and control.

Details on test conditions:

The medium used for the test was AAP-Medium (according to Annex 3 of OECD 201). The pH was adjusted to 7.5 r 0.1 with NaOH or HCl, if necessary.
Test Units
Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution
Test Conditions
Test procedure: Dose response test (static)
Duration: 72 hours Temperature: 22.6 – 23.0 °C
CO2 supplied: By continuous agitation: Test vessels were placed in an incubator on a pivoted bogie which turns around and induces shaking by regular sudden stops
pH of control: 7.29 – 8.33
Illumination: Continuously from the side, 88.8 µEm-2s-1 (mean)
Targeted initial cell density: 0.5 x 104 cells/mL
The incubation took place in a temperature controlled light incubator

Biological Assessment
The daily fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0). At defined dates (24, 48 and 72 hours), the number of cells in each replicate was determined in duplicate. The determination was performed by fluorescence measurement. By the means of a calibration curve, where fluorescence signals (y axis) were plotted versus cell numbers (x axis), the cell numbers were derived from the fluorescence signals. To establish a calibration curve, the cell numbers were counted with a Neubauer chamber after preparation of a dilution series of a logarithmic growing Pseudokirchneriella subcapitata culture.
Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.

Physico-Chemical Assessment
Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at hours 0, 24, 48 and 72. The light intensity of all positions of the incubator is measured once a year and was confirmed for one representative position at test start.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.54 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.:

32.2 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.:

3.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.05 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The measured initial concentration of guanylurea phosphate ranged from 88 % to 110 % of nominal. In the aged samples the measured content was between 95 % and 108 % of nominal. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item.

After 72 h at termination of the test statistically significant inhibition of growth rate was observed for test item concentrations of 9.77 mg/L and above. For yield statistically significant effects were observed at 3.05 mg/L and above. At higher test item concentrations the inhibition of growth rate peaked in 90.7 % and the inhibition of yield peaked in 99.3 % at a nominal test item concentration of 100 mg/L.

The morphology of the algae cells was observed microscopically at test end. The cells were considered morphologically normal for the control and up to and including a nominal test item concentration of 9.77 mg/L. No cells were observed at 31.3 mg/L test item concentration and above. The growth conditions (pH and temperature) during the test were within the range specified by OECD 201

Validity Criteria of the Study
Biomass Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 71.69 for the control, which exceeds the threshold of 16. It corresponds to a growth rate of 1.41840 d-1.
Coefficient of Variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 31 % for the control and did not exceed 35 %.
Coefficient of Variation (average growth):
The coefficient of variation of average growth in replicate control cultures was 4.3 % for the control and did not exceed 7 % for the whole test period.

The mean light intensity of all positions of the incubator was 88.8 µEm-2s-1 with mean values of each platform in a range from 82.6 to 93.6 µEm-2s-1 (-7.0 % to +5.4 % of mean) which was within ± 15 % of variation as specified by OECD 201.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

See table below.

1) Probit analysis following Gompertz distribution

2) Following Dunnetts-t-test (left-sided, p<0.05) 

3) Following Jonckheere Terpstra test (left sided, p<0.05)

Conc. (mg/L)	% inhibition of growth rate
	0 – 24h	0 – 48 h	0 – 72 h
Control	0.0	0.0	0.0
0.954	-3.1	5.5	3.3
3.05	-9.4	4.8	4.8
9.77	-8.6	11.4	15.1*
31.3	4.3	37.9	49.2*
100	83.5	75.9	90.7*

* Statistically significant different to the control

Validity criteria fulfilled:

yes

Conclusions:

Significant inhibitory effects were determined for growth rate at test item concentrations of 9.77 mg/L (nominal) and above. For yield significant inhibitory effects were determined at 3.05 mg/L (nominal) and above. The overall LOEC was therefore determined to be 3.05 mg/L (nominal), the corresponding NOEC was set at 0.954 mg/L (nominal). The EC10-value for growth rate (ErC10) was determined to be 5.54 mg/L (nominal). The EC10-value for yield (EyC10) was 0.964 mg/L (nominal). The EC20-value for growth rate (ErC20) was determined to be 11.2 mg/L (nominal). The EC20-value for yield (EyC20) was 2.35 mg/L (nominal). The EC50-value for growth rate (ErC50) was determined to be 32.2 mg/L (nominal). The EC50-value for yield (EyC50) was 8.99 mg/L (nominal).

Executive summary:

The study was performed according to OECD test guideline 201 (2011). Alage ( Pseudokirchneriella subcapitata) were exposed for 72 hours to test concentrations of 100, 31.3, 9.77, 3.05, 0.954 mg/L and control under static conditions. Six replicates were employed for the control and three for each test item concentration. Initial target cell densities of 0.5 × 104 cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start. Analytical samples were taken and analysed from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions.  

The validity critera of the test method were met.

Significant inhibitory effects were determined for growth rate at test item concentrations of 9.77 mg/L (nominal) and above. For yield significant inhibitory effects were determined at 3.05 mg/L (nominal) and above. The overall LOEC was therefore determined to be 3.05 mg/L (nominal), the corresponding NOEC was set at 0.954 mg/L (nominal). The EC10-value for growth rate (ErC10) was determined to be 5.54 mg/L (nominal). The EC10-value for yield (EyC10) was 0.964 mg/L (nominal). The EC20-value for growth rate (ErC20) was determined to be 11.2 mg/L (nominal). The EC20-value for yield (EyC20) was 2.35 mg/L (nominal). The EC50-value for growth rate (ErC50) was determined to be 32.2 mg/L (nominal). The EC50-value for yield (EyC50) was 8.99 mg/L (nominal).  

Description of key information

The study was performed according to OECD test guideline 201 (2011). Alage ( Pseudokirchneriella subcapitata) were exposed for 72 hours to test concentrations of 100, 31.3, 9.77, 3.05, 0.954 mg/L and control under static conditions. Six replicates were employed for the control and three for each test item concentration. Initial target cell densities of 0.5 × 104 cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. The pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily. Light intensity of the continuous illumination was measured at test start. Analytical samples were taken and analysed from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions.  

The validity critera of the test method were met.

Significant inhibitory effects were determined for growth rate at test item concentrations of 9.77 mg/L (nominal) and above. For yield significant inhibitory effects were determined at 3.05 mg/L (nominal) and above. The overall LOEC was therefore determined to be 3.05 mg/L (nominal), the corresponding NOEC was set at 0.954 mg/L (nominal). The EC10-value for growth rate (ErC10) was determined to be 5.54 mg/L (nominal). The EC10-value for yield (EyC10) was 0.964 mg/L (nominal). The EC20-value for growth rate (ErC20) was determined to be 11.2 mg/L (nominal). The EC20-value for yield (EyC20) was 2.35 mg/L (nominal). The EC50-value for growth rate (ErC50) was determined to be 32.2 mg/L (nominal). The EC50-value for yield (EyC50) was 8.99 mg/L (nominal).  

Key value for chemical safety assessment

EC50 for freshwater algae:

32.2 mg/L

EC10 or NOEC for freshwater algae:

3.05 mg/L