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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From December 18, 2017 to December 21, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Remarks:
To demonstrate that test vessels were dosed with nominal exposure concentrations of test substance, samples were analysed using the gas chromatography mass spectrometry method.
Details on sampling:
Samples for analysis at test start were taken from excess test solutions and at test end from a single test replicate solution from the control and each test concentration.
Vehicle:
yes
Details on test solutions:
Preparation of test solutions
The study was run with a culture medium control and nominal exposure concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L. Two primary stock concentrates of test substance, with nominal concentrations of 5 and 10 mg/L, were prepared. The 5 mg/L stock was prepared by adding a nominal 0.010 g of test substance (actual weight of 0.01011 g) and making up to 2000 mL volume with culture medium. The 10 mg/L stock was prepared by adding a nominal 0.010 g of test substance (actual weight of 0.01001 g) and making up to 1000 mL volume with culture medium. Both stocks were ultrasonicated for 100 minutes and assessed as homogenous dispersions with very fine particles visible. The 5 and 10 mg/L stocks were used directly as the 5 and 10 mg/L test solutions. The 5 mg/L stock was also used to prepare the remaining test solutions by direct addition of the appropriate amount to culture medium. The control consisted of culture medium only. In all cases the final solutions contained nutrients. The control, 0.625 and 1.25 mg/L test solutions were observed to be clear and colourless. The 2.5, 5 and 10 mg/L were observed to be homogenous dispersions with very fine particles visible. The appropriate test solution (100 mL volume) was dispensed to each test and blank vessel.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 3-d old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium and under the environmental conditions described for the test. The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22 ± 2ºC
pH:
7.20 to 7.71
Nominal and measured concentrations:
A 10 mg/L test concentration was selected as the highest concentration based on the dispersibility of the test substance demonstrated in a non-GLP solubility trial and the results of a non GLP range-finding study.
0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L (nominal)
0, 0, 0, 0, 0 and 0.78 mg/L (mean measured)
Details on test conditions:
Test appratus
The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 22  2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.78 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: Growth rate and biomass
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 0.78 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
other: Growth rate and biomass
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
> 0.78 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
> 0.78 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (total fraction)
Basis for effect:
biomass

Results

Analytical data

The limit of quantification (LOQ) of test substance in this study was 0.8 mg/L for all test solutions. The instrument LOQ was 0.4 mg/L but during analysis samples were diluted ×2, doubling the LOQ. Analytical calibrations were constructed using a minimum of 5 calibration levels, with a minimum R2 value of 0.995. The maximum percentage difference from nominal concentration for standards at the LOQ is less than 20% and less than 15% at levels greater tha the LOQ. All analytical values are quoted to two significant figures and percentages to the nearest integer. On the basis of the analytical data the mean measured concentrations were used for the calculation and reporting of results.

Biological data

Algal cell particle densities (cell particles per unit volume) were measured as a surrogate for biomass. The algal cell particle densities measured at each time period are given in Table 1.

Nominal

concentration of test substance

(mg/L)

Mean measured concentration of
test substance

(mg/L)

Replicate

Algal cell particle density

(×104cells/mL)

24 h

48 h

72 h

Culture medium control

0

A

1.67

6.34

33.30

B

1.47

5.59

31.20

C

1.51

7.42

34.80

D

1.63

6.41

37.20

E

1.63

7.02

33.00

F

1.49

6.96

31.80

Mean

1.57

6.62

33.55

0.625

0

A

1.55

7.21

33.70

B

1.67

6.90

31.30

C

1.70

7.26

31.40

Mean

1.64

7.12

32.13

1.25

0

A

1.69

7.43

31.50

B

1.59

7.46

35.30

C

1.59

6.61

31.60

Mean

1.62

7.17

32.80

2.5

0

A

1.77

6.63

31.90

B

1.68

6.29

27.40

C

1.72

8.14

33.60

Mean

1.72

7.02

30.97

5.0

0

A

1.77

6.34

27.50

B

1.94

6.20

31.00

C

2.19

7.03

32.60

Mean

1.97

6.52

30.37

10

0.78

A

2.15

7.18

29.80

B

2.29

5.53

29.20

C

2.71

5.81

29.50

Mean

2.38

6.17

29.50

Table 1: Algal cell particle density

 

Growth rates

The growth rate (0 to 72 h) was calculated for each replicate culture. The growth rates were examined by one-way analysis of variance and t-tests to identify significant differences (p <0.05) from the control. The EC50, EC20 and EC10 values with their associated confidence intervals were not calculated in this case due to the lack of biological response and analytical data.

The mean growth rates are given in Table 2, together with the rates expressed as percentages of the control.

Nominal concentration of test substance

(mg/L)

Mean measured concentration of test substance

(mg/L)

Mean growth rate / day

(0-72 h)

Mean growth

rate / day

95% Cl

Percentage of control (%)

Culture medium control

0

1.386

1.363 – 1.408

-

0.625

0

1.372

1.337 – 1.407

99

1.25

0

1.378

1.324 – 1.432

99

2.5

0

1.358

1.271 – 1.446

98

5.0

0

1.352

1.280 – 1.424

98

10

0.78

1.343

1.335 – 1.352

97

Table 2: mean growth rate over the test period

All values are quoted to 3 decimal places.

The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:

 

Test substance

(mg/L)

95% confidence limits

NOEC

0.78

-

LOEC

>0.78

-

ErC50

>0.78

n/a

 

Yield

This response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass. These cell particle densities were examined by one-way analysis of variance and t-tests to identify significant differences (p <0.05) from the control. The EC50, EC20 and EC10 values with their associated confidence intervals were not calculated in this case due to the lack of biological response and analytical data.

The yield mean values are shown in Table 3, together with the yields expressed as percentages of the control.

Nominal concentration of test substance

(mg/L)

Mean measured concentration of test substance

(mg/L)

Mean yield

(0-72 h)

(×104 cells/mL)

Percentage of control (%)

Culture medium control

0

33.0

-

 

0.625

0

31.6

96

1.25

0

32.3

98

2.5

0

30.4

92

5.0

0

29.8

90

10

0.78

29.0

88

Table 3: Mean yields over the test period

The results obtained from these statistical analysis, were as follows:

 

Test substance

(mg/L)

95% confidence limits

NOEC

0.78

-

LOEC

>0.78

-

EyC50

>0.78

n/a

 

Additional biological data

The microscopic observations, made at test end, showed that for the control and all test concentrations, the cells appeared normal.

Validity criteria

The validity criteria specified in the OECD 201 guideline are;

- To achieve ≥16 fold exponential increase in biomass in the control replicates within the 72 h test period. In this test, cell particle density increase (measured as a surrogate for biomass) was 64 times over the 72 h.

- The mean coefficients of variation for control replicates sectional (daily) specific growth rates must not exceed 35% and in this test, was determined to be 20.6% for the control.

- The replicate coefficient of variation of average specific growth rates during the whole test period in the control replicate cultures must not exceed 7% and was calculated as 1.5% for the control.

The study has met all validity criteria.

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of the read across study, 72 h ErC50, EyC50, NOEC and LOEC values for the test substance, mono- C16 PSE + C16-OH with freshwater green algae, were determined to be >0.78, >0.78, 0.78 and >0.78 mg/L, respectively.
Executive summary:

A study was conducted to determine the acute toxicity potential of the read across substance, 'mono- and di- C16 PSE, K+, H3PO4' (purity: 100%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 0.594 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.524 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L in AAP medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored using a GC/MS method of analysis. Based on whole sample extraction (dissolved and undissolved), the measured concentration of the test substances were found to be 0, 0, 0, 0, 0 and 0.78 mg/L. The test results were expressed in terms measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. ErC50 value (for growth rate) and EyC50 value (cell particle density) were determined to be >0.78 and >0.78 mg/L respectively. The NOEC and the LOEC values were found to be 0.78 mg/L and >0.78 mg/L, respectively, for both growth rate and cell particle densities. All validity criteria were met during this study. Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values were determined to be >0.78, >0.78, 0.78 and >0.78 mg/L, respectively (Scymaris, 2018). Based on the results of the read across study, similar EC50 and NOEC values can be expected for the test substance, 'mono- C16 PSE and C16 -OH'.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: DIN 38412-9: Determination of inhibitory effects of water constituents on the growth of Scenedesmus Green Algae
Version / remarks:
This method corresponds to the OECD Guideline 201
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Stock and test solution and preparation
Dispersion: none
Vehicle, solvent: None
Other procedures: test substance directly weighed into test vessels
Test organisms (species):
other: Scenedesmus subspicatus
Details on test organisms:
Strain: Scenedesmus subspicatus SAG 8681
Supplier: Institute of Plant Physiology, University of Gottingen
Photoperiod: continuous illumination
Intensity of irradiation: 2000 lux
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
22-23 deg C
Nominal and measured concentrations:
0, 10, 30, 100, 300 and 1000 mg/L (Nominal)
Details on test conditions:
Laboratory culture: not reported
Method of cultivation: not reported
Pretreatment: not reported
Controls: without test substance
Initial cell concentration: 1*10exp4 cells/mL
Renewal of test solution: none
Exposure vessel type: 300 mL Erlenmeyer flasks
Number of replicates: 3
The water solubility of hexadecanol is about 0.01 mg/L, therefore the no effect level appears to be above the saturation limit. The loading rates were all markedly above the solubility, which suggests that the dose-response is artefactual.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
other: EbL50
Effect conc.:
ca. 676 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
biomass
Remarks on result:
other: i.e., equivalent to 642.2 mg a.i./L
Key result
Duration:
96 h
Dose descriptor:
other: ErL50
Effect conc.:
> 980 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: i.e., equivalent to 931 mg a.i./L
Key result
Duration:
96 h
Dose descriptor:
other: ErL10
Effect conc.:
ca. 206 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: i.e., equivalent to 195.7 mg a.i./L
Key result
Duration:
96 h
Dose descriptor:
other: EbL10
Effect conc.:
ca. 24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
biomass
Remarks on result:
other: i.e., equivalent to 22.8 mg a.i./L
Details on results:
Results: Exposed
Nominal/measured concentrations: nominal

Effect data/Element values:
a) Growth rate
ErL0 = 98 mg/L, ErL10 = 206 mg/L, ErL50 = >980 mg/L
b) Biomass
EbL0 = 9.8 mg/L, EbL10 = 24 mg/L, EbL50 = 676 mg/L

Cell density data:
Cell densities increased from 3.7 - 4.3 * 10 exp 4 cells/mL after 24 h to the following densities after 96 h: 2.2 * 10 exp 6 (10 mg/L), 1.1 * 10 exp 6 (30 mg/L), 1.2 * 10 exp 6 (100 mg/L), 9 * 10 exp 5 (300 mg/L) and 9 * 10 exp 5 (1000 mg/L)

Results control:
Cell density increased from 2.7 * 10 exp 4 after 24 h to 2.1 * 10 exp 6 cells/mL after 96 h
Validity criteria fulfilled:
not specified
Conclusions:
Under study conditions, 96 h ErL50 and EyL50 values for the test substance with freshwater green algae, were determined to be >980 (i.e., 931 mg a.i./L) and 676 mg/L (i.e., 642.2 mg ai./L) (nominal), respectively.
Executive summary:

A study was conducted to determine the acute toxicity of the test substance, hexadecan-1-ol (purity: >95%), to the fresh water green algae (Scenedesmus subspicatus), according to DIN 38412-9, in compliance with GLP. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 10, 30, 100, 300 and 1000 mg/L in culture medium for 96 h. No analytical data was reported. The test results were expressed in terms of nominal concentration of test substance. Under study conditions, 96 h ErL50, (for growth rate) and EyL50 (biomass) values of the test substance for toxicity to freshwater green algae, were determined to be >980 (i.e., 931 mg a.i./L) and 676 mg/L (i.e., 642.2 mg ai./L) (nominal), respectively. The ErL10 and EbL10 values were determined to be 206 and 24 mg/L (i.e., 195.7 and 22.8 mg a.i./L) respectively. The water solubility of test substance is about 0.01 mg/L, therefore the no effect level can be considered to be above the saturation limit (OECD SIDS, 2006).

Description of key information

Based on the available weight of evidence from studies on the main constituents, similar absence of toxicity up to highest soluble test concentrations can be expected for the test substance, ‘mono- C16 PSE and C16-OH’ in freshwater green algae. Further, as a conservative approach, the lowest 72 h ErC50 and NOEC values of >0.78 mg/L and 0.78 mg/L (measured), has been considered further for hazard/risk assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.78 mg/L
EC10 or NOEC for freshwater algae:
0.78 mg/L

Additional information

In absence of specific study with the test substance, the toxicity to algae endpoint has been assessed based on studies for substances representative of the two main constituents, which can be categorised as phosphate esters (PSE) and alcohol. The results are presented below:

Constituent 1: PSE - read across study:

A study was conducted to determine the acute toxicity potential of the read across substance, ‘mono- and di- C16 PSE, K+, H3PO4’ (purity: 100%), to freshwater green algae (Pseudokirchneriella subcapitata), according to OECD Guideline 201, in compliance with GLP. Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. Each replicate test vessel was inoculated with 0.594 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.524 × 104 cells/mL. This value was used for growth calculations. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L in AAP medium for 72 h. The exposure levels of test substance in aqueous samples of test media were monitored at start (0 h) and end of the test (72 h), using a GC/MS method of analysis. Except for the measured values of the top test concentration at 0 h (i.e., determined to be 1.6 mg/L), the remaining measurements at 0 and 72 h were found to be below the limit of quantification (LOQ). Therefore, the test concentrations were presented as mean measured concentrations i.e., 0, 0, 0, 0, 0 and 0.78 mg/L. The test results were expressed in terms measured concentration of test substance. Algal cell particle densities (cell particles per unit volume measured as a surrogate for biomass) and growth rate were calculated for each replicate culture. ErC50 value (for growth rate) and EyC50 value (cell particle density) were determined to be >0.78 and >0.78 mg/L respectively. The NOEC and the LOEC values were found to be 0.78 mg/L and >0.78 mg/L, respectively, for both growth rate and cell particle densities. All validity criteria were met during this study. Under the study conditions, 72 h ErC50, EyC50, NOEC and LOEC values were determined to be >0.78, >0.78, 0.78 and >0.78 mg/L, respectively (Scymaris, 2018).

Constituent 2: Alcohol:

A study was conducted to determine the acute toxicity of hexadecan-1-ol (purity: >95%), to the freshwater green algae (Scenedesmus subspicatus), according to DIN 38412-9, in compliance with GLP. Three replicate algal cultures were exposed to test substance at nominal concentrations of 0, 10, 30, 100, 300 and 1000 mg/L in culture medium for 96 h. No analytical data was reported. The test results were expressed in terms of nominal concentration of test substance. Under study conditions, 96 h ErL50 (for growth rate) and EyL50 (biomass) values were determined to be >980 (i.e., 931 mg a.i./L) and 676 mg/L (i.e., 642.2 mg a.i./L) (nominal), respectively. The ErL10 and EbL10 values were determined to be 206 and 24 mg/L (i.e., 195.7 and 22.8 mg a.i./L) respectively. The water solubility of test substance is about 0.01 mg/L, therefore the no effect level can be considered to be above the saturation limit (OECD SIDS, 2006).

Based on the available weight of evidence from studies on the main constituents, similar absence of toxicity up to highest soluble test concentrations can be expected for the test substance, ‘mono- C16 PSE and C16-OH’ in freshwater green algae. Also, the presence of higher fatty alcohol content in the test substance is not found to have a differential impact on the acute toxicity potential in algae and it’s no effect level can be considered to be above the saturation limit of 0.01 mg/L. Further, as a conservative approach, the lowest 72 h ErC50 and NOEC values of >0.78 and 0.78 mg/L (measured), has been considered further for hazard/risk assessment.