Rhatany, Krameria triandra, ext.

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:

04.07.2018 - 05.12.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed in compliance with the Principle of Good Laboratory Practice, confirmed by Statement of GLP Compliance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

The following deviations were documented:
- The stock solutions to be autoclaved were autoclaved for 20 minutes instead of the required 15 minutes. This can be considered uncritical, as the goal of sterility is achieved.
- The temperature in experiment I was in the range 20.2 – 25.2°C, the temperature in experiment II was in the range 16.0 – 26.7°C and therefore not in the desired range (21.0 - 24.0°C). As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
- The temperature during pre-culture incubation for experiment I was in the range 15.1 – 26.1°C, the temperature in experiment II was in the range 23.8 – 27.7°C and therefore not within the desired range (21.0 - 24.0°C).. Because normal growth was observed, this can be stated as uncritical.
- The pre-culture was prepared 5 days before each test start. As all validity criteria were met and exponential growth was observed in the blank control this was stated as uncritical.
The deviations were assessed and signed by the study director on 13. Aug. 2018.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Rhatany, Krameria triandra, ext.

EC Number:

283-919-1

EC Name:

Rhatany, Krameria triandra, ext.

Cas Number:

84775-95-1

Molecular formula:

not available

IUPAC Name:

Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Krameria triandra, Krameriaceae.

Test material form:

liquid: viscous

Specific details on test material used for the study:

The following information concerning identity and composition of the test item was provided by the sponsor.
Name Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction
Batch no. PES180014
Appearance reddish brown viscous substance
Composition Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction
CAS No. 84775-95-1
EINECS-No. 283-919-1
Molecular weight not applicable, UVCB
Purity not applicable, UVCB
Homogeneity inhomogeneous, warm up to about 60°C and stir
Stability H2O: 96h; EtOH: 96h; acetone: 96h; CH3CN: 96h; DMSO: 96h
Solubility H2O: unknown; EtOH: >1 g/L; acetone: >1 g/L; CH3CN: >1 g/L; DMSO: >1 g/L
Production date Jan. 2018
Expiry date Jan. 2019
Storage Fridge (2 - 8 °C)

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

The test item was heated to about 60°C and stirred before use.
The water-accommodated fractions (WAFs) were prepared for the test. This was done by mixing the nominal loads (1 / 2.2 / 4.6 / 10 / 22 mg/L) with the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 23 hours. The resulting solution was filtrated through 0.45 µm PTFE filters.

Test solutions

Vehicle:

no

Details on test solutions:

All solutions were sterilised before use.
Composition of the Solutions:

Stock Solution I
NH4Cl 1500 mg
MgCl2*6H2O 1200 mg
CaCl2*2H2O 1800 mg
MgSO4*7H2O 1500 mg
KH2PO4 160 mg
H2O deionised ad 1000 mL

Stock Solution II
FeCl3*6H2O 64 mg
Na2EDTA*2H2O 100 mg
H2O deionised ad 1000 mL

Stock Solution III
H3BO3 185 mg
MnCl2*4H2O 415 mg
ZnCl2 3 mg
CoCl2*6H2O 1.5 mg
CuCl2*2H2O 0.01 mg
Na2MoO4*2H2O 7 mg
H2O deionised ad 1000 mL

Stock Solution IV
NaHCO3 50 g
H2O deionised ad 1000 mL

Algal medium
Stock solution I 10.0 mL
Stock solution II 1.0 mL
Stock solution III 1.0 mL
Stock solution IV 1.0 mL
H2O deionised ad 1000 mL

Stock solutions I and III and the deionised water were sterilized using an autoclave. Stock solutions II and IV were sterilized using filtration. The algal medium corresponds to the OECD TG 201 medium.
Deviations from the nominal weighted loads were less than 5%.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
Unicellular freshwater green alga.

Study design

Test type:

static

Water media type:

other: OECD TG 201 medium

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

16.0 – 26.7 °C

pH:

In the following table, the pH values measured at the start and the end of the test are stated:

Nominal Concentration in mg/L 0 h 72 h
Blank control 7.7 8.7
1 7.5 8.4
2.2 7.4 8.5
4.6 7.4 7.8
10 7.4 7.7
22 7.4 7.7

Details on test conditions:

Lighting: 5000 lux

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Basis for effect:

other: Yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 1 mg/L

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 1 mg/L

Basis for effect:

other: Yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.09 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.53 mg/L

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.12 mg/L

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.73 mg/L

Details on results:

In the following table, the appearance of the algae at the end of the test is stated:
Nominal Concentration in mg/L Normal and Healthy Appearance of the Algae
Blank control Yes
1 Yes
2.2 Yes
4.6 No cells visible
10 No cells visible
22 No cells visible

Results with reference substance (positive control):

see above

Reported statistics and error estimates:

All inhibition values were calculated by comparing the value for the endpoint of the treatment with the respective mean value of all blank controls (100%). Inhibition values are given in percent.

% Inhibition
Nominal concentration in mg/L Growth Rate (0-72h) Yield (0-72h)
Blank control 0 0
1 8.8 35.0
2.2 13.5 49.7
4.6 59.6 95.6
10* 71.29 97.89
22* 70.72 97.81

Calculation of results was performed with the help of validated software (Microsoft Excel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1.

Any other information on results incl. tables

The cell numbers were determined with an electronic particle counter; the details are given in chapter 16 Annex 2: Detailed Data. The means and standard deviations of the cell num-bers of the blank control and the treatments are presented in the following table:

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2400	11117	66680	374347
Blank control	SD	0	1123	3674	23784
1	Mean	2400	7593	45900	244207
1	SD	0	895	6800	53775
2.2	Mean	2400	6413	26367	189473
2.2	SD	0	855	752	5730
4.6	Mean	2400	5060	6520	18647
4.6	SD	0	413	314	3079
10	Mean	2400	6300	8287	10253
10	SD	0	140	602	926
22	Mean	2400	5820	9120	10560
22	SD	0	151	641	1081

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was performed using 5 concentrations ranging from 1 to 22 mg/L (see chapter 13 Deviations). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield1 were de-termined from the cell number at the respective observation times.
Significant inhibition of algal growth was observed at all relevant concentrations.
At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solu-tions using a carbon analyser.
Due to the low solubility the test item could be determined analytically only in the two high-est concentrations. The geometric mean can be determined from these measured values. For example, in the highest treatment (22 mg/L nominal) the geometric mean is 6.2 mg/L (based on a carbon content of 50.51%) and 7.4 mg/L (based on a carbon content of 42.39%). Different carbon contents were determined due to the inhomogeneity of the test item.
The lower concentrations were all in the same very low range as the blank control. How-ever, due to the inhibition values, the presence of the test item in the solutions can be as-sumed. Since each solution was prepared individually (and not diluted from a stock solu-tion) it is not possible to calculate back to the concentration of the low concentrations with the help of dilution factor.
According to the OECD guidance document no. 23, chapter 5, test substance that cannot be quantified by analytical methods at the effect concentrations, the biological results can be expressed based on the nominal concentrations. Since strong inhibition occurred at thethree highest concentrations and inhibition values above 50% were still achieved even with the 4.6 mg/L treatment, only the three lowest concentrations are used for statistical evalua-tion. The analytical results are nevertheless listed in the report.
72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).
The following results for the test item Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction were determined:
Endpoint NOEC LOEC EC10 EC50
Growth Rate < 1 mg/L ~ 1 mg/L 2.09 mg/L 4.12 mg/L
Yield < 1 mg/L ~ 1 mg/L 0.53 mg/L 1.73 mg/L
Note: According to the guideline, NOEC is determined by comparing of the respective treatment with the blank control. Statistically insignificant variation is considered as “no observed effect”, although the EC10 which is read from the graph toxicity vs. concentration may lie lower.

Executive summary:

This study was performed in order to evaluate the toxicity of Krameria triandra extract obtained from Rhatany root by hydroalcoholic extraction towards Desmodesmus subspicatus. This freshwater green alga was chosen as a typical species of phytoplankton. The system response was the reduction of growth in a series of algal cultures (test units) exposed to the test item. The response was evaluated as a function of the exposure concentration in comparison with the average growth of replicate, unexposed blank control cultures. For full expression of the system response to toxic effects (optimal sensitivity), the cultures were allowed unrestricted exponential growth under nutrient sufficient conditions and continuous light for a sufficient period of time to measure reduction of the specific growth rate. A positive control was tested in a separate study to assure that the test conditions are reliable.

Two experiments were performed. In the 1^stexperiment one of the validity criteria was not met (Mean coefficient of variation of daily growth rates). Therefore, the experiment was repeated. The results of the 1^stexperiment are not stated in the report, but will be kept together with the other raw data in the GLP archive of the test facility. The study was performed using 5 concentrations ranging from 1 to 22 mg/L (see chapter 13 Deviations). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield²were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at all relevant concentrations.

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser. Due to the low solubility the test item could be determined analytically only in the two highest concentrations. The geometric mean can be determined from these measured values. For example, in the highest treatment (22 mg/L nominal) the geometric mean is 6.2 mg/L (based on a carbon content of 50.51%) and 7.4 mg/L (based on a carbon content of 42.39%). Different carbon contents were determined due to the inhomogeneity of the test item. The lower concentrations were all in the same very low range as the blank control. However, due to the inhibition values, the presence of the test item in the solutions can be assumed. Since each solution was prepared individually (and not diluted from a stock solution) it is not possible to calculate back to the concentration of the low concentrations with the help of dilution factor.

According to the OECD guidance document no. 23, chapter 5, test substance that cannot be quantified by analytical methods at the effect concentrations, the biological results can be expressed based on the nominal concentrations. Since strong inhibition occurred at the three highest concentrations and inhibition values above 50% were still achieved even with the 4.6 mg/L treatment, only the three

lowest concentrations are used for statistical evaluation. The analytical results are nevertheless listed in the report.

The 72h-EC50values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory. The pH of the blank control should not fluctuate by more than 1.5 units. The change was 1.0 units in the blank control. All validity criteria were met. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.