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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with national standard methods with acceptable restrictions.
Qualifier:
according to guideline
Guideline:
other: US EPA (1978), The Selenastrum Capricornutum Printz Algal Assay Bottle Test, EPA 600/9-78-018.
Principles of method if other than guideline:
Method: other
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test media was prepared as described in EPA 600-78-018.
Standard solutions of the test substance were prepared in water and used to make a calibration curve that bracketed the expected stock concentration. Concentrations based on total test material were then adjusted to reflect the active acid component.
Stock solution was stored at room temperature in the dark.
Test type:
static
Water media type:
freshwater
Limit test:
no
Nominal and measured concentrations:
Nominal test concentrations used were 0.13, 0.74, 1.32, 7.4, 13.22, 74.04 and 132.22 mg active acid/L
Details on test conditions:
SOLVENT: None.
TEST APPARATUS: 500 ml flasks containg 100 ml of test medium. 
TEST DESIGN: Test performed in triplicate.
INITIAL INOCULUM: 10000 cells/ml. 
LIGHTING: 400 foot candles. 
GROWTH MEASUREMENT: Cell counts performed with a particle counter at 96 hours and every other day thereafter. Colorimetric determination was conducted using the Hach persulfate/UV oxidation method. Samples were analysed on a Perkin-Elmer-Hitachi 200 UV/visible spectrophotometer at 700nm.
WATER QUALITY: Not reported.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 132.22 mg/L
Nominal / measured:
nominal
Conc. based on:
other: active acid
Basis for effect:
growth rate
Remarks:
according to OECD TG 201 calculations
Remarks on result:
other: Interpreted by reviewer.
Remarks:
See "Any other information on results incl. tables" for reviewer interpretation.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
13.22 mg/L
Nominal / measured:
nominal
Conc. based on:
other: active acid
Basis for effect:
growth rate
Remarks:
according to OECD TG 201 calculations
Remarks on result:
other: Interpreted by reviewer
Remarks:
See "Any other information on results incl. tables" for reviewer interpretation
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
74.04 mg/L
Nominal / measured:
nominal
Conc. based on:
other: active acid
Basis for effect:
growth rate
Remarks:
according to OECD TG 201 calculations
Remarks on result:
other: Interpreted by reviewer
Remarks:
See "Any other information on results incl. tables" for reviewer interpretation
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
13 mg/L
Basis for effect:
growth rate
Remarks:
as reported in study report
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
39 mg/L
Basis for effect:
growth rate
Remarks:
as reported in study report
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks:
as reported in study report
Details on results:
- Exponential growth in the control (for algal test): yes over 96 hours, but not 14 days
- Observation of abnormalities (for algal test): no abnormalities reported
- Effect concentrations exceeding solubility of substance in test medium: no
Reported statistics and error estimates:
Reviewer has reinterpreted raw data to calculate EC50, NOEC and LOEC values in terms of growth rate according to OECD TG 201, and using RStudio Software (version 1.1.419).
NOEC and LOEC values were calculated for the effects of the substance on growth rate by analysing the specific growth rates for each replicate and applying a Levene's test for homogeneity of variance and Shapiro-Wilk test for normality of data. The data was normally distributed (p>0.05), but indicated heterogeneity of variance (p<0.05). Therefore, the growth rates were analysed by a one-way Welch's Anova for unequal variances test (RCompanion, 2015) and a Tukey's post hoc test, which demonstrated whether any of the test concentrations were statistically significantly different to the control.
RCompanion (2015) An R Companion for the Handbook of Biological Statistics. [Available at: https://rcompanion.org/rcompanion/d_05.html]

Table 1. Reinterpretation of data according to OECD TG 201 by study reviewer

Nominal concentration (mg active acid/L)

Day 0 cell concentration (cells/ml)

Day 4 cell concentration (cells/ml)

96h Average specific growth rate

Mean

SD

COV

% inhibition

control

10000

4085013

12.92025038

control

10000

4580853

13.03481069

control

10000

464447

10.74601764

control

10000

451715

10.71822164

11.85483

1.297282

10.94307

0.13

10000

4663760

13.05274745

0.13

10000

4544720

13.02689158

0.13

10000

4811389

13.08391128

13.05452

0.028551

0.218706

-10.1199

0.74

10000

3959093

12.88894042

0.74

10000

4881973

13.09847491

0.74

10000

3756349

12.83637294

12.94126

0.138664

1.071484

-9.16452

1.32

10000

3355653

12.72357185

1.32

10000

3629920

12.80213607

1.32

10000

4122186

12.92930907

12.81834

0.103821

0.809943

-8.12761

7.4

10000

563920

10.94008258

7.4

10000

580693

10.9693924

7.4

10000

505680

10.83107424

10.91352

0.072886

0.667846

7.9403

13.22

10000

491227

10.80207653

13.22

10000

423173

10.65295126

13.22

10000

464827

10.74683548

10.73395

0.075392

0.702374

9.454974

74.04

10000

8560

6.752270376

74.04

10000

8613

6.758442876

74.04

10000

8533

6.749111186

6.753275

0.004746

0.070281

43.03353

132.22

10000

22213

7.705847889

132.22

10000

20533

7.627203534

132.22

10000

21600

7.677863501

7.670305

0.039863

0.519709

35.29803

Result expressed as nominal concentration. Properties of the test substance and evidence from other studies (where
concentrations were measured) indicate that nominal and measured concentrations are likely to be in good agreement.

Table 2. Mean cell concentrations (cells/ml) after 96 hours and 14 days, as reported by study report
Nominal test concentration (mg/l)      
 96 hours        14 days
0                                       
                     4456859       5413760
0.13                                     
                   4673289       5679840
0.74                                     
                   4199137       5659875
1.32                                     
                   3702586       5821164
7.4                                      
                    550097       7281022
13.22                                  
                    459742       7311875
74.04                                  
                        8568       81458
132.22                                
                       21448       8622
             

96 hr EC50: 3 mg/l, 96 hr NOEC: 0.74 mg/l, as reported by study report. Toxicity decreased with time. At 14 days cultures exposed to 7.4 and 13.22 mg/l concentrations showed increased cell growth compared to controls. There is evidence that the cultures did not remain in exponential growth during the phase of the test extending from 96 hours to 14 days.

Validity criteria fulfilled:
not specified
Remarks:
Study predates OECD TG 201. Cell concentrations were not reported for any test concentration or control for 24 hours, 48 hours or 72 hours. However, the control replicates demonstrated specific growth rates of >0.92/day
Conclusions:
96-hour ErC50 and NOErC values of >132.22 and 13.22 mg active acid/L, respectively, were determined by the study reviewer for the effects of HEDP-H on the growth rate of Pseudokirchneriella subcapitata (reported as: Selenastrum capricornutum).
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to Annex 3 of the CSR and IUCLID Section 13 for justification of read-across between members of the HEDP category.
Reason / purpose for cross-reference:
read-across source
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 132.22 mg/L
Nominal / measured:
nominal
Conc. based on:
other: active acid
Basis for effect:
growth rate
Remarks:
according to OECD TG 201 calculations
Remarks on result:
other: Interpreted by reviewer
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
13.22 mg/L
Nominal / measured:
nominal
Conc. based on:
other: active acid
Basis for effect:
growth rate
Remarks:
according to OECD TG 201
Remarks on result:
other: Interpreted by reviewer
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
74.04 mg/L
Nominal / measured:
nominal
Conc. based on:
other: active acid
Basis for effect:
growth rate
Remarks:
according to OECD TG 201
Remarks on result:
other: Interpreted by reviewer

Description of key information

96-hour ErC50 >132.22 mg active acid/L and NOErC 13.22 mg active acid/L, Pseudokirchneriella subcapitata, read-across from HEDP-H.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
13.22 mg/L

Additional information

A 96-hour ErC50 value of >132.22 mg active acid/L and a NOErC value of 13.22 mg active acid/L have been determined for the effects of HEDP-H on the growth rate of Pseudokirchneriella subcapitata (SRI International, 1980). These values have been calculated by the study reviewer, according to the calculations of average specific growth rate in the OECD TG document 201. The study reported a 96-hour EbC50 value of 3 mg active acid/L based on biomass and 14-day NOEC and LOEC values of 13 mg active acid/L and 39 mg active acid/L, respectively, based on growth rate. However, the EbC50 value is based on biomass and the control group did not demonstrate exponential growth over the 14-day exposure period. Therefore, the values calculated by the study reviewer are selected as key for this endpoint as they represent a standard exposure duration in which exponential growth was maintained in the control group.

Several supporting algal studies of reliability rating 4, are available with HEDP acid and its salts. Three results are available for the registration substance, HEDP (2-3Na): a 14-day LOEC value of 30 mg/L was determined for the effects on the cell number of Selenastrum sp. and a 48 -day EC50 value of >960 mg/L was determined for the effects on the cell number of Chlorella vulgaris (Henkel, 1984a and b). Two 18-day NOEC values of 10, 10 and <10 mg/L have been determined for the effects of HEDP-H on the biomass of Chlorella sp., Anabaena sp. and Selenastrum sp., respectively (Monsanto, 1972a, b and c).

A reliable study reported 96-hour EC50 values of 12, 2.5 and 8.8 mg active acid/L for the effects of HEDP-xNH4 on the cell number of P. subcapitata in: normal test media conditions, test media with added calcium and test media with added calcium and magnesium conditions, respectively. NOEC values of 50, <6.2 and <6.2 mg active acid/L were reported for the effects of HEDP-xNH4 on the cell number of P. subcapitata under the same respective test media conditions (Springborn, 1992).

A supporting study is available that reports EC50 values of 7.23 mg/L, 14.96 mg/L and 0.07 mg/L for the effects of HEDP-H on the biomass of P. subcapitata in test media and hard water, however it was assigned a reliability rating of 3, based on the reduction in pH, which decreased to 3.11 in the highest test concentration (Monsanto, 1992).

The effects of HEDP observed in tests with algae are likely to be a consequence of nutrient availability limitations caused by complexation and not true toxicity (see the discussion below). The test results that are available cannot therefore be taken into account to assess the toxicity of HEDP (2-3Na). They have been included as supporting information to show the variability in determined EC50 and NOEC values and to illustrate the issues with testing substances that exhibit chelating behaviour. The available evidence suggests that toxic effects observed in the tests are a consequence of complexation of essential nutrients and not of true toxicity.

The acid, sodium and potassium salts in the HEDP category are freely soluble in water and, therefore, the HEDP anion is fully dissociated from its sodium or potassium cations when in solution. Under any given conditions, the degree of ionisation of the HEDP species is determined by the pH of the solution. At a specific pH, the degree of ionisation is the same regardless of whether the starting material was HEDP-H, HEDP (1-2Na), HEDP (2-3Na), HEDP-4Na, HEDP-xK or another salt of HEDP.

 

Therefore, when a salt of HEDP is introduced into test media or the environment, the following is present (separately):

  1. HEDP is present as HEDP-H or one of its ionised forms. The degree of ionisation depends upon the pH of the system and not whether HEDP (1-2Na), HEDP (2-3Na), HEDP-4Na, HEDP-xK salts, HEDP-H or another salt was added.
  2. Disassociated sodium/potassium cations. The amount of sodium/potassium present depends on which salt was added.
  3. Divalent and trivalent cations have much higher stability constants for binding with HEDP than the sodium or potassium ions, so would preferentially replace them. These ions include calcium (Ca2+), magnesium (Mg2+) and iron (Fe3+). Therefore, the presence of these in the environment or in biological fluids or from dietary sources would result in the formation of HEDP-dication (e.g. HEDP-Ca, HEDP-Mg) and HEDP-trication (e.g. HEDP-Fe) complexes in solution, irrespective of the starting substance/test material.

In this context, for the purpose of this assessment, read-across of data within the HEDP Category is considered to be valid.

Nutrient complexation in algal test medium  

It is a functional property of phosphonate substances that they form stable complexes (ligands) with metal ions. In algal toxicity tests essential nutrients will thus be bound to the phosphonates according to the Ligand binding model[1]. In algal growth medium some metals form strongly-bound complexes and others form weakly-bound ones. The phosphonates possess multiple metal-binding capacities, and pH will affect the number of binding sites by altering the ionisation state of the substance. However, the phosphonate ionisation is extensive regardless of the presence of metals (Girling et al. 2010).

The phosphonate-metal complexes may be very stable due to the formation of ring structures ("chelation"). This behaviour ensures that the phosphonic acids effectively bind and hold the metals in solution and renders them biologically less available As a result when a trace metal is complexed, its bioavailability is likely to be negligible (Girling et al. 2010, SIAR 2004). However, there is no evidence of severe toxicity from metal complexes of the ligands (Girling et al. 2010).

In algal growth inhibition tests, complexation of essential trace nutrients (including Fe, Cu, Co, and Zn) by phosphonate substances can lead to inhibition of cell reproduction and growth. Guidelines for toxicity tests with algae do not typically describe procedures for mitigating against this behaviour. For example the standard OECD Guideline 201, describing the algal growth inhibition test, only specifies that the “chelator content” should be below 1 mmol/l in order to maintain acceptable micronutrient concentrations in the test medium (SIAR 2004).

OECD guidance on the testing of difficult substances and mixtures (OECD, 2000) does include an annex describing “toxicity mitigation testing with algae for chemicals which form complexes with and/or chelate polyvalent metals”. The procedure is designed to determine whether it is the toxicity of the substance or the secondary effects of complexation that is responsible for any observed inhibition of growth. It involves testing the substance in its standard form and as its calcium salt in both standard algal growth medium and in medium with elevated CaCO3 hardness. Calcium is non-toxic to aquatic organisms and does not therefore influence the result of the test other than by competitively inhibiting the complexation of nutrients (SIAR 2004). By increasing the calcium content it may be that the nutrient metals are released from their complexed form although this may not always apply. The outcome of the test however only determines whether nutrient complexation is the cause of apparent toxicity and does not determine the inherent toxicity of the test substance for the reasons explained by the ligand binding model (Girling et al. 2018).

The magnitude of the stability constants depends on the properties of the metal and also of the ligand, in respect of the type of bonding, the three dimensional shape of the complexing molecule, and the number of complexing groups. The SIAR provides two tables of stability constants (effectively the strength of the complexation), one from Lacour et al. (1999) and one from Gledhill and Feijtel (1992). The Gledhill and Feijtel constants show a range of values for important divalent metal ions, cited as having been obtained from Monsanto internal reports (Owens, 1980). They show that ATMP, HEDP and DTPMP are strong complexing agents, with stability constant values ranging from 6 to 24 (Log10 values), as presented in the following table.


Table: Stability constants of phosphonates.

Type

CAS Number

Ca

Cd

Co

Cu

Hg

Mg

Ni

Pb

Zn

Fe

ATMP

6419-19-8

7.6

12.7

18.4

17

21.7

6.7

15.5

16.4

14.1

approximately 25a

ATMP-N-oxide-H

15834-10-3

5.69

No data

No data

No data

No data

8.29

No data

No data

No data

approximately 25a

BHMT

34690-00-1

6.1b

12b

18b

20b

22b

6.3b

20b

13b

19b

approximately 25a

DTPMP-H

15827-60-8

6.7

9.7

17.3

19.5

22.6

6.6

19

8.6

19.1

approximately 25a

HEDP

2809-21-4

6.8

15.8

17.3

18.7

16.9

6.2

15.8

No data

16.7

approximately 25a

HMDTMP-H

23605-74-5

5.4

13.3

18.9

19.8

21.9

6

20.6

17

19.5

approximately 25a

EDTMP-H

1429-50-1

9.6

21.4

15.8

24.3

31.8

10

20.2

23

21.1

approximately 25a

Notes:

a - The complexation constant for phosphonates with iron (III) has been estimated by TNO (1996a) to be around log K = 25.

b – In the absence of experimental data, the stability constants of BHMT complexes has been estimated as the mean of the stability constants for each metal ion as measured with the structural analogues DTPMP and HMDTMP.


 

The complexation constant for phosphonates with iron (III) has been estimated by TNO (1996a) to be around log K = 25 (Girling et al. 2010).

All the algal toxicity studies available for phosphonates that have used standard and non-standard test conditions are presented in Girling et al. (2010). The studies show a large variation of toxicity for these substances sharing similar physico-chemical properties, with reliable EC50 varying from 0.1 to 450 mg/l.

The most refined study to date is the DTPMP study undertaken by TNO laboratories (1996) where concentrations of Cu, Co and Zn, were increased in the medium in line with their complexation strength (Cu up to 30 times, Co up to 30 times and Zn up to 300 times). When Fe was also added up to 300 times the guideline concentration no toxic effects were seen at the highest tested concentration (96h ErC50 equivalent to >10 mg/L). The increased amounts of Fe meant that complex iron-DTPMP bonds were formed, leaving the four nutrients free for algal uptake. The test demonstrates effects of iron-DTPMP complex to algae, not any effects of the free substance. The media concentration of Fe in the study is a highly unlikely scenario in a true environmental exposure, where Ca and Mg are likely to be more readily available but are also more weakly complexed. Where essential nutrients with stronger binding capacity are present, such as Cu, Co, Zn and Fe, the phosphonates will preferentially bind to these nutrients leaving the Ca and Mg free.

In Springborn Laboratories (1992) the mitigation procedures suggested in the OECD guidance on testing difficult substances (2000) were adopted when testing with HEDP acid (CAS 2809-21-4). The authors increased water hardness, complexed the test substance with CaCl2 and additionally performed a standard test which achieved 96-hour EC50 values of 8.8, 3.5 and 12 mg/l respectively based on cell numbers. While the results are contrasting, the test does not reflect the true toxicity of the test substance since essential nutrients such as Co and Fe will, according to the ligand binding model and stability constants, continue to be preferentially bound and thus not be bioavailable to the algae. In the same manner results of a test carried out by HLS (2001) with elevated nutrient levels (x25 times) to counterbalance nutrient complexation by DTPMP-xNa (CAS 22042-96-2), will not be representative of inherent toxicity since the amounts of essential nutrients added will not be enough to counteract the phosphonates’ Fe and Co preferential complexation and as a result the nutrients will remain unavailable, inhibiting cell multiplication.

In addition SRI International (1984) tested the effects of EDTMP acid with a diatom and two species of cyanobacteria while increasing the nutrients in the test medium (x0.5 to x3 standard nutrient concentrations) to counteract the complexing effects of phosphonates. The general trend in the results supports that it is nutrient complexation that is the cause of the effects seen in the studies.The available evidence suggests that toxic effects observed in the tests are a consequence of complexation of essential nutrients and not of true toxicity. A study designed to ensure adequate levels of bioavailable nutrients with either of the phosphonates would result in the test substance being a phosphonates-Fe complex. Under conditions where iron is readily available to counteract the effects of nutrient complexation it is unlikely that the substance would have a negative effect on algal growth (Girling et al. 2010). The nutrient complexing behaviour of phosphonate substances therefore renders testing to determine their intrinsic toxicity to algae impractical.

Prolonged (14-day) studies show a decrease in toxicity with time. For example SRI International (1981) reports a 96-hour ErC50 value of 0.42 and a 14-day ErC50 value of 27 mg/l when testing EDTMP acid with Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata) under standard conditions. This mitigation of effects adds to the evidence that it is not inherent toxicity that is causing the observed effects. This is thought to be attributable to the release of phosphorous by the gradual photodegradation of the phosphonic substances.

The interpretation of these data is also consistent with findings presented in the risk assessment being carried out for the chelating agent EDTA (CAS 60-00-4, Risk Assessment 2004), which is actually a weaker complexing agent than BHMT. It has been demonstrated that for EDTA it is not the absolute concentration, but rather the ratio of the EDTA concentration to that of the metal cations that is crucial to determining algal growth under the conditions of a toxicity test (EC, 2003).

The ability of iron to catalyse photodegradation of phosphonates means that the interpretation of all algal growth data is somewhat uncertain; this applies to the complexing agents discussed above including EDTA. However, limitation of micronutrient availability is considered to be a sufficiently generic phenomenon to explain effects observed in toxicity tests with substances that have the capacity to chelate cationic metals (Girling et al. 2010).

Available data on effects to algae and aquatic plants have been reviewed and discussed in the peer-reviewed and published SIAR (please refer to Section 4.1.3 of the SIAR). The conclusion(s) or critical result(s) from the SIAR are as follows:

A total of nine results from tests with three freshwater genera were available for consideration - two results from short-term (96-hour) tests and seven results from prolonged-term tests (14 to 18 days). None of the tests satisfied the requirements for achieving a reliability rating of 1 but two short-term and two prolonged-term tests were of an acceptable standard for assessing the toxicity of the substance. A reliable short-term (96-hour) test with Selenastrum capricornutum yielded an EC50, based on growth rate, of 3.0 mg/L. The lowest reliable NOEC determined in the prolonged tests was 13 mg/L (14-day), although there is evidence that the cultures did not remain in exponential growth during the phase of the test extending from 96 hours to 14 days. A 14-day LOEC of 1-10 mg/L and a 21-day NOEC of 3 mg/L were also determined in other tests, the reliability of which could not be assessed.

A detailed interpretation of the effects of nutrient complexation by, and photolytic release of phosphorus from, phosphonic acids on algal growth in toxicity studies is given in Annex V to the phosphonic acid SIARs (2004). The principle conclusions of the review are that:

·        Algal growth may be stimulated by the presence of supplementary phosphorous released by the photolytic degradation of phosphonic acids.

·        Algal growth may be inhibited by the complexation of micronutrients (trace metals) by phosphonic acids. This inhibition is an algistatic rather than algicidal effect. Under the standard test conditions used for most studies, the trace metals will be fully and strongly bound to the HEDP, with the strong possibility that their bioavailability will have been reduced considerably.

These two phenomena can occur at different stages in the course of the same algal test and at different exposure levels of the substance.

The ability of iron to catalyse photodegradation of phosphonates means that the interpretation of algal growth data can be somewhat uncertain; this applies to the complexing agents discussed above including EDTA. However, limitation of micronutrient availability is considered to be a sufficiently generic phenomenon to explain effects observed in toxicity tests with substances that have the capacity to chelate cationic metals.

Conclusions: Great care has to be exercised in interpreting the results of the algal tests carried out with phosphonic acids. The significant potential for nutrient complexation by phosphonates and/or release of phosphorous from degradation of phosphonates to respectively either inhibit or stimulate algal growth makes definitive interpretation difficult. However the available evidence suggests that toxic effects observed in tests with structurally analogous substances are a consequence of complexation of essential nutrients and not of true toxicity. These effects do not obey a classic dose response and as such extrapolation using an assessment factor is inappropriate. In addition, similar effects would not be anticipated in natural environmental waters. Therefore further algal toxicity studies are not recommended.

Please see the attached position paper (Girling et al, 2018) which further discusses algal tests with phosphonate substances and presents arguments against further algal testing.


[1] 'Ligand’ is a general term used to describe a molecule that bonds to a metal; in the present case the phosphonate can form several bonds and the resultant chelated complex can be a very stable entity. It is possible that two molecules could bind to the individual metal, or that one molecule could bind two metals. In dilute solution a 1:1 interaction is the most probable. To simplify discussion, the ligand is considered to be able to form a strongly-bound complex with some metals, and a more weakly-bound complex with others.