Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine

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:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental study performed according to the OECD 201 guideline and under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Please, see TMI above

Analytical monitoring:

yes

Details on sampling:

The analytical samples were sampled at the beginning of the test in the test solutions preparations and at the end of the test in the content of the vessels pooled by replicate.
The following protocol was applied:
- 1 mL of sample was collected and pipetted into a 2 mL micro-centrifugation tube;
- For all samples, 1 mL was collected with a syringe and was filtered (Filter Macherey Nagel RC-45/13 ref 729237) into a 1.5 mL glass amber vial.

Details on test solutions:

Test item was supplied by the sponsor as a saturated solution. Aqueous phase of this solution was used as Limit of Solubility solution (LS). As this saturated solution was prepared with deionised water, stock solutions of media were added at LS solution as below:
Test item solution (LS) = 500 mL
Media stock solution (SM1) = 5 mL
Media stock solution (SM2) = 0.5 mL
Media stock solution (SM3) = 0.5 mL
Media stock solution (SM4) = 0.5 mL
Final volume (F) = 506.5 mL
Dilution factor (F / LS) = 1.013 mL
Dilution factor (1.013) was negligible to performed the test at LS concentration.
The test solution was then prepared by dilution of test item solution

Test organisms (species):

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

Details on test organisms:

The Pseudokirchneriella subcapitata stock culture was maintained at 4°C and regularly transferred to fresh medium at the laboratory. The algal stock culture was provided by the CCAP (Culture Collection of Algae and Protozoa), reference CCAP 278/4. A culture was prepared from the stock culture and maintained in an exponential growth by successive transfers to fresh medium used in the test (OECD medium) every 3 to 4 days.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Hardness:

Not specified

Test temperature:

21.7 to 23.1 °C (mean: 22.1°C)

pH:

7.6 - 8.1

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 0; LS/16; LS/8; LS/4; LS/2 and LS
Measured (T0): 0; 0.16, 0.33, 0.65, 1.31 and 2.61 mg/L
Measured (72h): 0; 0.17, 0.33, 0.65, 1.31 and 2.60 mg/L

Details on test conditions:

Based on the results of the no-GLP range-finding test, the test solutions were prepared in OECD medium at the definitive nominal concentrations of LS/16; LS/8; LS/4; LS/2 and LS. A control was performed at the same time with test media only.
Every flask was inoculated with the initial cell concentration of 5*10^3 cells.mL-1 of Pseudokirchneriella subcapitata as recommended in the guideline OECD 201.

Starting from a culture of 3.55E+06 cells per millilitres, 705 µL were inoculated in 500 mL of OECD medium for the control (C02) and 352.5 µL were inoculated in 250 mL of OECD medium for the tested concentrations (C03 to C07), to reach a concentration of about 5000 cells per milliliters. The preparations were divided in 6 replicates for the control and 3 replicates for the tested concentrations consisting in about 80 mL per replicates.
The flasks consisted of glass Erlenmeyer flasks of 250 mL capacity, closed with air-permeable stoppers made of polyether foam.
The test was performed in a thermostatic chamber maintained in the range of 21°C to 24°C controlled to ± 2°C under continuous illumination (with a light intensity between 4440 and 8880 lux). Algae were maintained suspended under continuous orbital stirring.
According to the study plan, cell biomass was measured every day in each flask using an electronic particle counter.
At the end of the test, after 72 hours of exposure, a microscopic observation was performed to verify a normal and healthy appearance of the inoculum culture and to observe any abnormal appearance of the algae.
pH was recorded at the beginning and the end of the test in all treatments.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.61 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC50 was greater than the water solubility of the substance

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 2.61 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC10 was greater than the water solubility limit of the substance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.61 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

See the section "any information on results incl. tables".
No abnormal appearance of the algae at the start and at the end of the test was recorded.

Results with reference substance (positive control):

Potassium dichromate: EC-72h-50 (mg.L-1): 1.09
95% confidence limits (mg.L-1): 0.95 – 1.23

Table 1: Cells number on control flasks

	Number of cells at 0 h	Measure at 24 h (cells / mL)	Measure at 48 h (cells / mL)	Measure at 72 h (cells / mL)
Control C02	5000	29608	206600	1474000
	5000	27024	209100	1146000
	5000	23256	172300	1211000
	5000	18792	170100	1073000
	5000	14384	159700	1277000
	5000	29384	139000	1104000
Mean	5000	23741	176133	1214167

 

Table 2: Specific growth rates, growth rates and yields of control replicates

	Specific growth rate 0 h – 24 h	Specific growth rate 24 h – 48 h	Specific growth rate 48 h – 72 h	Mean of specific rate per replicate	Standard deviation of specific rate	CV of specific rate	Growth rate (day^-1) after 72h	CV growth 72h (%)	Yield (cells / mL) after 72h	CV yield 72h (%)
Control C02	1.779	1.943	1.965	1.895	0.102	5.370	1.895	2.1	1469000	12.2
	1.687	2.046	1.701	1.812	0.203	11.220	1.812		1141000
	1.537	2.003	1.950	1.830	0.255	13.931	1.830		1206000
	1.324	2.203	1.842	1.790	0.442	24.687	1.790		1068000
	1.057	2.407	2.079	1.848	0.704	38.122	1.848		1272000
	1.771	1.554	2.072	1.799	0.260	14.465	1.799		1099000
Mean	1.526	2.026	1.935	1.829	0.328	17.966	1.829		1209167

Table 3: Cells number, growth rates and yields in the different replicates of the test item group

Rates	Number of cells at 0 h	Measure at 24 h (cells / mL)	Measure at 48 h (cells / mL)	Measure at 72 h (cells / mL)	Growth rate (day^-1) after 72h	CV growth 72h (%)	Growth rate inhibition (%) compared to control after 72h	Yield (cells / mL) after 72h	CV yield 72h (%)	Yield inhibition (%) compared to control after 72h
mg test item/L after 72h
LS/16 (C03)	5000	31264	129800	1086000	1.794	0.5	1.93	1081000	3.0	10.60
	5000	32800	144500	1024000	1.774		3.00	1019000		15.73
	5000	26336	176200	1052000	1.783		2.51	1047000		13.41
Mean	5000	30133	150167	1054000	1.784		2.48 (ns)	1049000		13.25 (ns)
LS/8 (C04)	5000	19336	198300	906900	1.734	0.8	5.21	901900	4.4	25.41
	5000	29288	168200	860900	1.716		6.16	855900		29.22
	5000	29648	180900	938800	1.745		4.58	933800		22.77
Mean	5000	26091	182467	902200	1.732		5.32 (*)	897200		25.80 (*)
LS/4 (C05)	5000	29984	194800	1040000	1.779	2.6	2.72	1035000	13.6	14.40
	5000	32376	195800	1282000	1.849		-1.10	1277000		-5.61
	5000	29576	155800	1359000	1.868		-2.16	1354000		-11.98
Mean	5000	30645	182133	1227000	1.832		-0.18 (ns)	1222000		-1.06 (ns)
LS/2 (C06)	5000	25496	194900	977400	1.758	2.9	3.85	972400	16.1	19.58
	5000	31456	198100	1312000	1.857		-1.52	1307000		-8.09
	5000	23584	166500	1042000	1.780		2.68	1037000		14.24
Mean	5000	26845	186500	1110467	1.798		1.67 (ns)	1105467		8.58 (ns)
LS (C07)	5000	30280	109700	968000	1.755	1.4	4.02	963000	7.5	20.36
	5000	28760	171600	902000	1.732		5.31	897000		25.82
	5000	23912	128300	1047000	1.781		2.59	1042000		13.82
Mean	5000	27651	136533	972333	1.756		3.98 (ns)	967333		20.00 (ns)

ns: not statistically significant compared to the control (p>0.05, t-test)

* statistically significant compared to the control (p<0.05, t-test)

 

Table 4: pH measurements

	C02	C03	C04	C05	C06	C07
pH at 0 h	7.7	7.7	7.7	7.7	7.7	8.1
pH at 72 h	7.6	7.6	7.6	7.6	7.6	7.9

 

Table 5: Temperature and light intensity during the test

Time	Mean	Min	Max
Temperature (TMP_0104)	22.1 °C	21.7 °C	23.1 °C
Light intensity (lux)	8023

Table 6: Validity criteria

	Actual value	Reference value		Compliance
Growth multiplication factor in the control within the 72-hour test period	243	>	16	Y
Coefficient of variation of the average specific growth rates in the control during the whole test period	2.12	<	7	Y
Mean coefficient of variation for section-by-section specific growth rates (days 0-1. 1-2 and 2-3) in the control	17.97	<	35	Y

Table 7: Analytical results

ID	Concentrations	0h(mg.L^-1)	72h(mg.L^-1)
C02	0	0	0
C03	LS/16	0.16	0.17
C04	LS/8	0.33	0.33
C05	LS/4	0.65	0.65
C06	LS/2	1.31	1.31
C07	LS	2.61	2.60

Validity criteria fulfilled:

yes

Conclusions:

HMX was not acutely toxic to Pseudokirchneriella subcapitata when tested at the solubility limit of the compound. ErC50 and ErC10 based on growth rate could not be determined because there were above the solubility limit of the substance, i.e. 2.61 mg/L.
The lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) were > 2.61 and 2.61 mg/L, respectively.

Executive summary:

The objective of this study was to assess the effects of test item Octogen solution on the growth of Pseudokirchneriella subcapitata over a period of 72 hours according to OECD 201 Guideline. Pseudokirchneriella subcapitata is a freshwater green algae that is recommended by the OECD Guideline 201.

The test item is a saturated solution supplied by the sponsor. The test solutions were prepared in the test medium by dilution of saturated test item solution, and were expressed in limit of solubility (LS). 

The effects of Octogen solution at the definitive nominal concentrations of 0; LS/16; LS/8; LS/4; LS/2 and LS to Pseudokirchneriella subcapitata were investigated under laboratory conditions.

The analytical samples were sampled at the beginning of the test and at the end of the test (72 hours) with the content of the vessels pooled and homogenized.

Since the deviation between the measured concentrations at T0 and the measured concentrations at 72h was within the range of ± 20 %, analysis of the results were based in the measured concentrations at T0.

The EC₁₀, EC₂₀, EC₅₀ and NOEC/LOEC values are summarized in the table below:

	Octogen solution
NOEC (growth rate and yield)	2.61 mg.L^-1
LOEC (growth rate and yield)	>2.61 mg.L^-1
72h-ErC10 (95% confidence limits)	>2.61 mg.L^-1
72h-ErC20 (95% confidence limits)	>2.61 mg.L^-1
72h-ErC50 (95% confidence limits)	>2.61 mg.L^-1
72h-EyC10 (95% confidence limits)	2.53 mg.L^-1(95% confidence limits: 0.80 – 51.90 mg.L^-1)
72h-EyC20 (95% confidence limits)	>2.61 mg.L^-1
72h-EyC50 (95% confidence limits)	>2.61 mg.L^-1

EC = Effect concentration

NOEC= No Observed Effect Concentration

LOEC = Lowest Observed Effect Concentration

Description of key information

For that endpoint, an experimental study was available to assess the effects of test item on the growth of Pseudokirchneriella subcapitata over a period of 72 hours according to OECD 201 Guideline and under GLP conditions.

Since the deviation between the measured concentrations at T0 and the measured concentrations at 72h was within the range of ± 20 %, analysis of the results were based in the measured concentrations at T0.

HMX was not acutely toxic to Pseudokirchneriella subcapitata when tested at the solubility limit of the compound. ErC50 and ErC10 based on growth rate could were above the solubility limit of the substance, i.e. 2.61 mg/L. The lowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) were > 2.61 and 2.61 mg/L, respectively.

All validity criteria were met, therefore this study was considered valid.

Additional information

For that endpoint, other data on the registered substance were available.

Indeed, the effect of HMX to Algae was assessed under static conditions in two freshwater static algae assays. One was a bottle test with four species of algae, the other was a microplate-based bioassay. Both studies were performed at the limit of solubility of HMX.

The 96h EC50 of test substance to Scenedesmus capricornutum is > 22 μmol/L (6.5 mg/L)  (Biotechnology Research Institute, 1997). The 96h-EC50 of the test substance to algae (Microcystis aeruginosa, Anabeana flos-aquae, Selenastrum capricornutum and Navicula pelliculosa) was greater than 32 mg/L (nominal concentration) (EG & G Bionomics, 1978).

Nevertheless, the key parameter investigated was the inhibition of growth after 96 hours (i.e. 96h-EC50) based on yield rather than growth rate (logarithmic increase in biomass). Therefore these studies do not provide an adequate coverage of the key parameter foreseen to be investigated in the corresponding test methods referred to in Article 13(3). In addition, no data is available on the analytical monitoring of exposure concentrations. Therefore these studies do not provide a reliable coverage of the reported key parameter. Finally, no data on section-by-section specific growth rates are available. Therefore these published studies do not provide adequate documentation to verify whether all validity criteria of the test methods referred to in Article 13(3) were fulfilled. In particular, the documentation is insufficient to verify if:

- the biomass in the control cultures increased exponentially by a factor of at least 16 within the 72-hour test period (i.e. specific growth rate ≥ 0.92 /day);

- the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, 2-3 and 3-4, for 96-hour tests) in the control cultures did not exceed 35%.

For these reasons, these two study have been finally disregarded.