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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May 2018 to 16 Jul 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Bis-Aminopropyl Diglycol Dimaleate
Appearance: Clear to transparent yellow liquid
Purity/Composition: Not indicated
Test item storage: At room temperature
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix 6).
Vehicle:
no
Details on test solutions:
The batch of Bis-Aminopropyl Diglycol Dimaleate tested was a clear to transparent yellow liquid of which the exact purity was unknown. The test item had a water content of 74% and was completely soluble in test medium at the concentrations tested. A correction factor of 3.85 was used and all further concentrations reported are expressed as the active ingredient (a.i.).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Stock culture
Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Light intensity
60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.


Stock culture medium
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture
Four days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium
M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
21 24°C, constant within ±1°C
pH:
6-9, not varying by more than 1.5 units
Nominal and measured concentrations:
Nominally 0.32, 1.0, 3.2, 10, 32 and 100 mg a.i./L
Time Weighted Average (TWA) concentrations were calculated to be 0.13, 0.39, 1.4, 4.3, 29 and 115 mg a.i/L.
Details on test conditions:
Test Procedure and Conditions:
Test duration : 72 hours
Test type: Static
Test vessels: 100 mL silanized all-glass vessels with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
Test Medium: M2
Cell densit: An initial cell density of 1 x 10^4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 72 to 76 µE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Measurements and Recordings:
pH: At the beginning and at the end of the test, for all test concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the final test microscopic observations were performed on the test concentration of nominally 32 mg a.i./L to observe for any abnormal appearance of the algae compared to the control.

Recording of Cell Densities:
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
96 other: ma a.i./L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% confidence levels: 85 to 108
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
39 other: ma a.i./L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other:
Remarks:
95% confidence levels: 35 to 44
Details on results:
See "Any other information on results, incl. tables".
Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ERC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.30 mg/L with a 95% confidence interval ranging from 0.29 to 0.31 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L.
Reported statistics and error estimates:
The study met the acceptability criteria prescribed by the study plan and was considered valid.

Mean Cell Densities, Inhibition of Growth Rate and Inhibition of Yield

A concentration-related increase of algal growth rate and yield inhibition was recorded at all test concentrations, reaching 46% growth rate and 90% yield inhibition at the limit concentration.

Based on these results, samples taken from nominally 0.10 and 100 mg a.i./L were analysed.In each sample, two test item related peaks were recorded that were identified as a cationic and an anionic constituent.Actual test item concentrations were expressed using either the measured cationic or anionic responses.

At the start of the test, actual test item concentrations were at the level of nominal (i.e. 92‑104% relative to nominal), based on the separate responses of both the cationic and anionic constituent. Actual test item concentrations based on the cationic constituent remained stable (i.e. 86-103% relative to nominal) throughout the test. Contrary, actual test item concentrations based on the anionic constituent decreased to 0.54-1.8% during the 72-hour exposure period (see also Table 3 and 5 of the appended Analytical Report).

All test conditions were maintained within the limits prescribed by the study plan.

Table 1          
Mean Cell Densities (x104Cells/mL) during the Combined Limit/Range-Finding Test


Time (h) 

Bis-Aminopropyl Diglycol Dimaleate
Nominal conc.
(mg a.i./L)

Control

0.10

1.0

10

100

0

1.0

1.0

1.0

1.0

1.0

24

4.4

n.d.

n.d.

n.d.

1.6

48

27

n.d.

n.d.

n.d.

6.9

72

149

133

131

109

15

n.d. – not determined

Table 2          
Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test


Bis-Aminopropyl Diglycol Dimaleate
Nominal conc.
(mg a.i./L) 

Mean

Std. Dev.

n

%Inhibition

Control

1.666

0.0204

6

 

0.10

1.629

0.0209

3

2.3

1.0

1.624

0.0143

3

2.5

10

1.562

0.0084

3

6.3

100

0.903

0.0764

6

46

  

Table 3          
Percentage Inhibition of Yield during the Combined Limit/Range-Finding Test


Bis-Aminopropyl Diglycol Dimaleate
Nominal conc.
(mg a.i./L) 

Mean

Std. Dev.

n

%Inhibition

Control

147.5

9.20

6

 

0.10

131.6

8.18

3

11

1.0

129.6

5.57

3

12

10

107.5

2.74

3

27

100

14.4

3.78

6

90

Final Test

Measured Test Item Concentrations

Samples taken from all test concentrations and the control were analysed. In each sample, two test item related peaks were recorded that were identified as a cationic and an anionic constituent. Similar to the combined limit/range-finding test, actual test item concentrations were expressed using either the measured cationic or anionic responses.

At the start of the test, the actual exposure concentrations were at the level of nominal (i.e. 99‑109% relative to nominal), based on the separate responses of both the anionic and cationic constituent. Based on the cationic constituent, the actual test item concentrations remained stable (i.e. 98-104% relative to nominal) throughout the test. Contrary, the actual test item concentrations based on the anionic constituent were at 0.65‑121% of nominal at the end of the test. It should be noted that a small response of both constituents was observed in the control which was, however, negligible compared to the responses recorded at any test concentration.

Similar to the combined limit/range-finding test, the pH of the test solutions at the start of the test decreased with increasing test item concentrations (seeTable 9). Hence, it is most likely that the test item affected the pH of the test medium. In addition, stability of the anionic constituent increased with increasing test item concentrations (see appended Analytical Report) and seemed to correlate to the pH. However, existence of this correlation cannot be proven and the exact reason for the difference in stability of the anion is unknown. An effect on the test item concentrations due to the presence of the algae can be excluded.For both constituents, the concentrations measured in the samples taken from solutions with algae were comparable to the concentrations measured in the samples without algae.

It is unknown which constituent caused toxicity towards algae. Therefore, effect parameters were based on the actual exposure concentrations determined from measurements of the anionic constituent as the worst-case scenario. Since test item concentrations were not stable during the exposure period, effect parameters were expressed in terms of Time Weighted Average (TWA) concentrations (seeTable 4).

 

Table 4          
Time Weighted Average Versus Nominal Concentrations


Test item1
Nominal conc. (mg a.i./L)

Measured concentration2(mg a.i./L)

TWA (mg a.i./L)

t=0h

t=24h

t=72 h

0.32

0.336

0.313

0.002563

0.13

1.0

1.08

0.973

0.006463

0.39

3.2

3.45

3.31

0.05693

1.4

10

10.4

11.1

0.1083

4.3

32

33.8

37.6

18.3

29

100

109

114

121

115

1Bis-Aminopropyl Diglycol Dimaleate,2test item concentration based on the recorded responses of the anionic constituent,3response was below the Lowest Calibration Standard (i.e. 0.1 mg/L) and therefore estimated by extrapolation of the calibration curve.

    

Mean Cell Densities

Figure 1 (attached below) shows growth curves from the control and the different TWA concentrations of Bis‑Aminopropyl Diglycol Dimaleate.

Inhibition of Growth Rate and Inhibition of Yield

Statistically significant inhibition of growth rate was found at TWA concentrations of 29 and 115 mg a.i./L. However, growth rate inhibition was considered to be biologically not relevant at the TWA concentration of 29 mg a.i./L at which the observed inhibition was below 10%. The NOEC based on biological relevance was thus set at 29 mg a.i./L.

Inhibition of yield increased with increasing concentrations of Bis-Aminopropyl Diglycol Dimaleate at the three highest test concentrations. Statistically significant inhibition of yield was found at TWA concentrations of 4.3 mg a.i./L and higher. However, yield inhibition was considered to be biologically not relevant at the TWA concentration of 4.3 mg a.i./L at which the observed inhibition was below 10%. The NOEC based on biological relevance was thus set at 4.3 mg a.i./L.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the TWA concentration of 32 mg a.i./L when compared to the control.

Table 5          
Growth Rate and Percentage Inhibition for the Total Test Period


Bis-Aminopropyl Diglycol Dimaleate
Nominal conc.
(mg a.i./L) 

Mean

Std. Dev.

n

%Inhibition

Control

1.521

0.0257

6

 

0.32 [0.13]

1.531

0.0149

3

-0.67

1.0 [0.39]

1.522

0.0215

3

-0.076

3.2 [1.4]

1.538

0.0144

3

-1.2

10 [4.3]

1.492

0.0292

3

1.9

32 [29]

1.381

0.0055

3

9.2#

100 [115]

0.638

0.1798

3

58*

* - effect was statistically significant,#- effect was statistically significant however biologically not relevant (<10%), [ ] – TWA concentration (mg a.i./L).

Table 6          
Growth Rate and Percentage Inhibition at Different Time Intervals


Test item1

Nominal conc. (mg a.i./L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

%Inhibition

Mean

%Inhibition

Mean

%Inhibition

Control

6

1.261

 

1.565

 

1.735

 

0.32 [0.13]

3

1.265

-0.31

1.459

6.8

1.868

-7.7

1.0 [0.39]

3

1.312

-4.0

1.431

8.6

1.822

-5.0

3.2 [1.4]

3

1.637

-30

1.112

29

1.865

-7.5

10 [4.3]

3

1.316

-4.3

1.417

9.5

1.742

-0.42

32 [29]

3

1.042

17

1.311

16

1.790

-3.2

100 [115]

3

0.455

64

0.382

76

1.077

38

1Bis-Aminopropyl Diglycol Dimaleate, [ ] – TWA concentration (mg a.i./L).

Table 7          
Yield and Percentage Inhibition for the Total Test Period


Bis-Aminopropyl Diglycol Dimaleate
Nominal conc.
(mg a.i./L) 

Mean

Std. Dev.

n

%Inhibition

Control

95.0

7.24

6

 

0.32 [0.13]

97.8

4.43

3

-2.9

1.0 [0.39]

95.2

6.08

3

-0.24

3.2 [1.4]

100.0

4.33

3

-5.3

10 [4.3]

87.1

7.81

3

8.3#

32 [29]

62.0

1.05

3

35*

100 [115]

6.5

3.99

3

93*

* - effect was statistically significant,#- effect was statistically significant but biologically not relevant (<10%), [ ] – TWA concentration (mg a.i./L).

 

Determination of Effect Concentration

Table 8 shows the effect parameters based on TWA concentrations, see also Appendix 4.

Table 8          
Effect Parameters


Parameter (mg a.i./L)

NOEC*

NOEC#

EC10

EC20

EC50

Growth rate

Value

4.3

29

30

45

96

lower 95%-cl

 

 

21

34

85

upper 95%-cl

 

 

39

54

108

Yield

Value

1.4

4.3

15

21

39

lower 95%-cl

 

 

11

17

35

upper 95%-cl

 

 

18

24

44

cl – confidence limit, * - based on statistical significance,#- based on biological relevance.

Experimental Conditions

The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit).

During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the study plan (21‑24°C, constant within ±1°C).

Table 9          
pH Levels Recorded during the Final Test


Bis-Aminopropyl Diglycol Dimaleate

Nominal conc.(mg a.i./L)

pH

t=0h

t=72h

Control

8.2

8.2

0.32 [0.13]

8.1

8.2

1.0 [0.39]

8.0

8.2

3.2 [1.4]

7.9

8.2

10 [4.3]

7.7

8.3

32 [29]

7.0

8.2

100 [115]

6.1

6.1

[ ] – TWA concentration (mg a.i./L).

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Bis-Aminopropyl Diglycol Dimaleate inhibited growth rate of this fresh water algae species significantly at Time Weighted Average (TWA) concentrations of 29 mg a.i./L and higher. Yield was inhibited significantly at TWA concentrations of 4.3 mg a.i/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was 96 mg a.i./L with a 95% confidence interval ranging from 85 to 108 mg/L.

The 72h-EC50 for yield inhibition (EYC50) was 39 mg a.i./L with a 95% confidence interval ranging from 35 to 44 mg/L.

The 72h-NOEC for growth rate inhibition was 4.3 mg a.i./L based on statistical significance.

The 72h-NOEC for growth rate inhibition was 29 mg a.i./L based on biological relevance.

The 72h-NOEC for yield inhibition was 1.4 mg a.i./L based on statistical significance.

The 72h-NOEC for yield inhibition was 4.3 mg a.i./L based on biological relevance.

It should be noted that all effect parameters as described above, are based on the TWA concentrations calculated from the responses of the anionic constituent as a worst-case scenario.
Executive summary:

The objective of the study was to evaluate Bis-Aminopropyl Diglycol Dimaleate for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20and EC50for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000. Furthermore, the study was performed under GLP conditions.

The batch of Bis-Aminopropyl Diglycol Dimaleate tested was a clear to transparent yellow liquid of which the exact purity was unknown. The test item had a water content of 74% andwas completely soluble in test medium at the concentrations tested.A correction factor of 3.85 was used and all further concentrations reported are expressed as the active ingredient (a.i.).

A final test was performed based on the results of a preceding combined limit/range-finding test.Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominally 0.32, 1.0, 3.2, 10, 32 and 100 mg a.i./L. The initial algal cell density was 104cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

At the start of the test, the actual exposure concentrations were at the level of nominal (i.e. 99‑109% relative to nominal), based on the separate responses of both the anionic and cationic constituent. Based on the cationic constituent, the actual test item concentrations remained stable (i.e. 98-104% relative to nominal) throughout the test. Contrary, the actual test item concentrations based on the anionic constituent were at 0.65‑121% of nominal at the end of the test. As a worst-case scenario, actual test item concentrations based on the anionic constituent were used to express effect parameters. Time Weighted Average (TWA) concentrations were calculated to be 0.13, 0.39, 1.4, 4.3, 29 and 115 mg a.i/L.

A concentration-related increase of both growth rate and yield inhibition was observed at concentrations of 4.3 mg a.i/L and higher.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg a.i./L)

NOEC*

NOEC#

EC10

EC20

EC50

Growth rate

Value

4.3

29

30

45

96

lower 95%-cl

 

 

21

34

85

upper 95%-cl

 

 

39

54

108

Yield

Value

1.4

4.3

15

21

39

lower 95%-cl

 

 

11

17

35

upper 95%-cl

 

 

18

24

44

cl – confidence limit, * - based on statistical significance,#- based on biological relevance.

Description of key information

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Bis-Aminopropyl Diglycol Dimaleate inhibited growth rate of this fresh water algae species significantly at Time Weighted Average (TWA) concentrations of 29 mg a.i./L and higher. Yield was inhibited significantly at TWA concentrations of 4.3 mg a.i/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was 96 mg a.i./L with a 95% confidence interval ranging from 85 to 108 mg/L.

The 72h-EC50 for yield inhibition (EYC50) was 39 mg a.i./L with a 95% confidence interval ranging from 35 to 44 mg/L.

The 72h-NOEC for growth rate inhibition was 4.3 mg a.i./L based on statistical significance.

The 72h-NOEC for growth rate inhibition was 29 mg a.i./L based on biological relevance.

The 72h-NOEC for yield inhibition was 1.4 mg a.i./L based on statistical significance.

The 72h-NOEC for yield inhibition was 4.3 mg a.i./L based on biological relevance.

It should be noted that all effect parameters as described above, are based on the TWA concentrations calculated from the responses of the anionic constituent as a worst-case scenario.

Key value for chemical safety assessment

EC50 for freshwater algae:
96 mg/L

Additional information